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WO2024020587A2 - Pleiopluripotent stem cell programmable gene insertion - Google Patents

Pleiopluripotent stem cell programmable gene insertion Download PDF

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Publication number
WO2024020587A2
WO2024020587A2 PCT/US2023/070781 US2023070781W WO2024020587A2 WO 2024020587 A2 WO2024020587 A2 WO 2024020587A2 US 2023070781 W US2023070781 W US 2023070781W WO 2024020587 A2 WO2024020587 A2 WO 2024020587A2
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Prior art keywords
cell
pleiopluripotent
locus
site
hla
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PCT/US2023/070781
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French (fr)
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WO2024020587A3 (en
Inventor
Jonathan Douglas FINN
Minggang FANG
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Tome Biosciences, Inc.
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Publication of WO2024020587A2 publication Critical patent/WO2024020587A2/en
Publication of WO2024020587A3 publication Critical patent/WO2024020587A3/en

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Definitions

  • this disclosure features a method of generating a pleiopluripotent cell, where the method includes site-specifically incorporating at least a first integration recognition site (e.g., AttB) into the genome of a pleiopluripotent cell, where site-specifically incorporating the genome of a pluripotent cell is effected by introducing into the pleiopluripotent stem cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein or a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the pleiopluripotent stem cell genome at sites respectively flanking the specific incorporation site, and at least one of the first paired guide RNAs is an atgRNA that further includes a
  • the first of the two paired guide RNAs is a first atgRNA that further includes a first RT template that comprises at least a portion of the first integration recognition site; and a second of the two paired guide RNAs is a second atgRNA that further includes a second RT template that comprises at least a portion of the first integration recognition site, wherein the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the method includes integrating at least a first donor polynucleotide template into the pleiopluripotent cell genome at the first incorporated integration recognition site, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the at least one incorporated genomic integration recognition site by the integrase; thereby producing a second generation pleiopluripotent cell.
  • this disclosure features a pleiopluripotent cell comprising at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome.
  • this disclosure features a pleiopluripotent cell comprising a donor polynucleotide template comprised of one or more orthogonal integration recognition sites integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • this disclosure features a pleiopluripotent cell or a population thereof, where (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site incorporated site-specifically into the PC- derived cell genome. In another aspect, this disclosure features.
  • this disclosure features a pleiopluripotent cell or a population thereof, wherein (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC- derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • the present disclosure provides nucleic acid compositions, methods, and an overall platform for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE) (see Ionnidi et al.; Nat. Biotech.41: 500–512 (2023)); the entirety of which is incorporated herein by reference), transposon-mediated gene editing, or other suitable gene editing, or gene incorporation technology.
  • PASTE Programmable Addition via Site-Specific Targeting Elements
  • this disclosure features a method of generating a pleiopluripotent cell, the method comprising: (a) site-specifically incorporating at least a first integration recognition site into a genome of a pleiopluripotent cell.
  • site-specifically incorporating the at least first integration recognition site is effected by introducing into the pleiopluripotent cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein or a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the pleiopluripotent cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site-specifically incorporated into the genome of the pleiopluripo
  • RT reverse transcripta
  • the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is a first atgRNA that further includes a first RT template that comprises at least a portion of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a second atgRNA that further includes a second RT template that comprises at least a portion of the first integration recognition site, wherein the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the at least first pair of guide RNAs comprise: [0014] (i) the first of the two paired guide RNAs is an atgRNA that further includes an RT template that comprises at least a portion of the first integration recognition site, wherein the atgRNA encodes the entirety of the first integration recognition site; and [0015] (ii) a second of the two paired guide RNAs is a nicking gRNA. [0016] In some embodiments, the method further comprises incorporating a plurality of integration recognition sites.
  • the method further comprises: (b) integrating at least a first donor polynucleotide template into the pleiopluripotent cell genome at the first incorporated integration recognition site, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a second generation pleiopluripotent cell.
  • this disclosure features a method of generating a pleiopluripotent cell, the method comprising: (a) integrating, into the genome of any of the pleiopluripotent cells described herein at the first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a second generation pleiopluripotent cell.
  • the method further comprises (b) site-specifically incorporating a second integration recognition site into the genome of the pleiopluripotent cell, thereby generating a third generation pleiopluripotent cell.
  • the method further comprises: (c) integrating a second donor polynucleotide template into the pleiopluripotent cell genome at the second incorporated integration recognition site, by delivering into the cell: (i) the second donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration recognition sites orthogonal to the second integration recognition site, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the second incorporated genomic integration recognition sites by the integrase; thereby producing a fourth generation pleiopluripotent cell line.
  • steps (a), (b), and (c) are performed concurrently.
  • step (a) is performed prior to steps (b) and (c), wherein steps (b) and (c) are performed concurrently or step (b) is performed prior to step (c).
  • step of site-specifically incorporating the first integration recognition site and the step of site-specifically incorporating the second integration recognition site are performed concurrently.
  • the first RT template encodes a first single-stranded DNA sequence and the second RT template encodes a second single-stranded DNA sequence.
  • the first single-stranded DNA sequence comprises a complementary region with the first single-stranded DNA sequence.
  • the first single-stranded DNA sequence and the first single-stranded DNA sequence form a duplex.
  • the complementary region is 5 or more consecutive bases.
  • the complementary region is 10 or more consecutive cases.
  • the complementary region is 20 or more consecutive bases.
  • the complementary region is 30 or more consecutive bases.
  • at least one of the two paired guide RNAs has a chemical modification.
  • the paired guide RNAs each have a chemical modification.
  • at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a safe harbor locus.
  • at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell.
  • At least one of the at least first integration recognition sites is incorporated into the genome at a plurality of loci, wherein disruption of at least one of the loci is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell.
  • at least one of the at least first integration recognition sites is incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
  • the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
  • the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
  • the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus.
  • the locus is the B2M locus.
  • the locus is the CIITA locus.
  • the introducing step is performed by electroporation.
  • introducing comprises electroporating a gene editor protein or a polynucleotide encoding a gene editor protein, a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein, at least a first pair or guide RNAs, the donor polynucleotide template, or a combination thereof into the pleiopluripotent cell occurs using one or more of a recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, anellovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, mRNA, RNP, or lipid nanoparticle.
  • the introducing step is performed by transfection.
  • introducing comprises transfecting mRNA encoding the gene editor protein, the prime editor fusion protein, the at least first pair of guide gRNAs, the donor polynucleotide template, or a combination thereof into the pleiopluripotent cell, wherein the donor polynucleotide template is selected from a mini circle, a nanoplasmid, and a miniDNA.
  • at least one of the at least first integration recognition sites is specific for a serine integrase.
  • At least one of the at least first integration recognition sites is an attB or attP site. [0044] In some embodiments of the method, at least one of the at least first integration recognition sites is a modified attB or attP site. [0045] In some embodiments of the method, at least one of the at least first integration recognition sites is specific for BxB1 or a modified BxB1. [0046] In some embodiments of the method, at least one of the at least first integration recognition sites is comprised of 38 or 46 nucleotides.
  • the first donor polynucleotide template encodes one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof.
  • the inducing the at least one of the one or more inducible suicide genes targets the pleiopluripotent cell or a cell matured from the pleiopluripotent cell for cell death.
  • expression of at least of the one or more inducible suicide gene is driven by a pluripotent stem cell-specific promoter.
  • expression of at least one of the one or more inducible suicide gene is driven by a constitutive promoter. In some embodiments of the method, expression of at least one of the one or more inducible suicide gene is driven by an inducible promoter.
  • the one or more inducible suicide genes is selected from: caspase9, cytosine deaminase, and thymidine kinase. In some embodiments of the method, the one or more inducible suicide genes is a controllable caspase9. In some embodiments of the method, AP20187 (or an analog thereof) controls activity of Caspase9 or AP21967 (or an analog thereof) controls activity of Caspase9.
  • the method further comprises a second inducible suicide gene, wherein the second inducible suicide gene comprises a thymidine kinase.
  • the polynucleotide encoding for the thymidine kinase is operably linked to the controllable capase9 protein using a 2A self-cleaving peptide or an internal ribosome entry site.
  • the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells.
  • the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells by inhibiting, blocking, or preventing recognition of the pleiopluripotent cell by macrophages, T- cells, Natural Killer (NK)-cells, antigen-presenting cells, or a combination thereof.
  • the one or more exogenous polypeptides capable of modulating an immune response are capable of serving as a safety switch target.
  • expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a pluripotent stem cell-specific promoter.
  • expression of least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a constitutive promoter.
  • expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by an inducible promoter.
  • the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL-1, B2M-HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR.
  • the donor polynucleotide template encodes a CD47 polypeptide, a PDL1 polypeptide, and a B2M-HLA-E polypeptide.
  • each of the sequences coding for CD47, PDL1 and B2M-HLA-E are separated by a sequence coding for a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof.
  • the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag.
  • the donor polynucleotide template further comprises an additional orthogonal integrase target recognition site.
  • the second integration recognition site is site- specifically incorporated into a safe harbor locus.
  • the second integration recognition site is different from the at least first integration recognition attB or attP site.
  • the second integration recognition site is specific for BxB1 or a modified BxB1.
  • the second integration recognition sites is comprised of 38 or 46 nucleotides.
  • a second donor polynucleotide template is integrated into the pleiopluripotent cell genome at the second integration recognition site site-specifically incorporated into the pleiopluripotent cell genome.
  • the second donor polynucleotide template encodes one or more therapeutic agents.
  • the one or more therapeutic agents is selected from: transcription factors, receptors, signaling molecules, secreted proteins, cytokines, enzymes, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, and persistence and/or survival of the iPSCs or cells derived from the iPSC.
  • one or more therapeutic agents is a HLA class I protein.
  • the one or more therapeutic agents is a HLA class II protein.
  • the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents.
  • the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agents.
  • the second donor polynucleotide template comprises an inducible promoter operably linked to at least one of the one or more therapeutic agents.
  • the first donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 1 kilobase (kb), at least 2 kb, at least 3kb, at least4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28k
  • the first donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 30kb.
  • the second donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 1 kilobase (kb), at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at
  • the second donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 30kb.
  • the pleiopluripotent cell is a pluripotent stem cell.
  • the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
  • the pluripotent stem cell is an induced pluripotent stem cell.
  • the induced pluripotent stem cell is a human induced pluripotent stem cell.
  • the method further comprises site-specifically excising from the genome of the pleiopluripotent cell the first donor polynucleotide template, the second donor polynucleotide template, or both.
  • the site-specifically excising is effected by introducing into the pleiopluripotent cell an integrase that recognizes the one or more orthogonal integration recognition sites in the first donor polynucleotide template, the second donor polynucleotide template, or both.
  • the method further comprises cryopreserving the pleiopluripotent cell or a population thereof.
  • this disclosure features a pleiopluripotent cell or population thereof generated using the method any of the methods described herein. [0086] In another aspect, this disclosure features a second generation pleiopluripotent cell generated using the method of any of methods described herein. [0087] In another aspect, this disclosure features a third generation pleiopluripotent cell generated using the method of any of the methods described herein. [0088] In another aspect, this disclosure features a fourth generation pleiopluripotent cell generated using the method of any of the methods described herein.
  • this disclosure features a pleiopluripotent cell, comprising: [0090] at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome.
  • this disclosure features a pleiopluripotent cell comprising: [0092] a donor polynucleotide template comprised of one or more orthogonal integration recognition site integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • the pleiopluripotent cell wherein at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a safe harbor locus.
  • at least one of the at least first integration recognition sites is site-specifically incorporated within a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell.
  • the pleiopluripotent cell at least one of the at least first integration recognition sites is site-specifically incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
  • the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
  • the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
  • the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA- DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus.
  • the locus is the B2M locus.
  • the locus is the CIITA locus.
  • At least one of the at least first integration recognition sites is specific for a serine integrase.
  • at least one of the at least first integration recognition sites is an attB or attP site.
  • at least one of the at least first integration recognition sites is a modified attB or attP site.
  • at least one of the at least first integration recognition sites is specific for BxB1 or a modified BxB1.
  • the pleiopluripotent cell at least one of the at least first integration recognition site is comprised of 38 or 46 nucleotides.
  • the donor polynucleotide template encodes one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof.
  • inducing at least one of the one or more inducible suicide genes targets the pleiopluripotent cell or a cell matured from the pleiopluripotent cell for cell death.
  • expression of at least one of the one or more inducible suicide gene is driven by a pluripotent stem cell-specific promoter. In some embodiments, expression of at least one of the one or more inducible suicide gene is driven by a constitutive promoter. In some embodiments, expression of at least one of the one or more inducible suicide gene is driven by an inducible promoter. In some embodiments, the one or more inducible suicide genes is selected from: caspase9, cytosine deaminase, and thymidine kinase. In some embodiments, the one or more inducible suicide genes is a controllable caspase9.
  • the pleiopluripotent cell further comprises a second inducible suicide gene.
  • the second inducible suicide gene comprises a thymidine kinase.
  • the polynucleotide encoding for the thymidine kinase is operably linked to the controllable capase9 protein using a 2A self-cleaving peptide or an internal ribosome entry site.
  • the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells. In some embodiments, the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells by inhibiting, blocking, or preventing recognition of the PC-derived cell by macrophages, T-cells, Natural Killer (NK)-cells, antigen-presenting cells, or a combination thereof. [0108] In some embodiments of the pleiopluripotent cell, the one or more exogenous polypeptides capable of modulating an immune response are capable of serving as a safety switch target.
  • pleiopluripotent cell expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a pluripotent stem cell-specific promoter. [0110] In some embodiments of the pleiopluripotent cell, expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a constitutive promoter. [0111] In some embodiments of the pleiopluripotent cell, expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by an inducible promoter.
  • the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL-1, B2M-HLA-E, HLA- G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof.
  • the donor polynucleotide template encodes a CD47, PDL1, and B2M-HLA- E.
  • the polynucleotide template encoding CD47, PDL1 and B2M-HLA-E are separated by a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof.
  • the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag.
  • the tag is an ALFA tag.
  • the pleiopluripotent cell comprises two integration recognition sites, three integration recognition sites, four integration recognition sites, five integration recognition sites, six integration recognition sites, seven integration recognition sites, eight integration recognition sites, nine integration recognition sites, or ten or more integration recognition sites.
  • the pleiopluripotent cell further comprises a second integration recognition site site-specifically incorporated into the pleiopluripotent cell genome.
  • the second integration recognition sites is site-specifically incorporated into a safe harbor locus.
  • the second integration recognition site is site-specifically incorporated within a second locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell.
  • the second integration recognition site is site-specifically incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
  • the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
  • the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
  • the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus.
  • the locus is the B2M locus. In some embodiments, the locus is the CIITA locus. [0116] In some embodiments of the pleiopluripotent cell, at least one of the at least second integration recognition sites is specific for a serine integrase. [0117] In some embodiments of the pleiopluripotent cell, the second integration recognition sites is different from the at least first integration recognition attB or attP site. [0118] In some embodiments of the pleiopluripotent cell, the second integration recognition sites is a modified attB or attP site.
  • the second integration recognition site is specific for BxB1 or a modified BxB1.
  • the second integration recognition sites is comprised of 38 or 46 nucleotides.
  • the second donor polynucleotide template encodes one or more therapeutic agents.
  • the one or more therapeutic agents is selected from: transcription factors, receptors, signaling molecules, secreted proteins, cytokines, enzymes, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, and persistence and/or survival of the iPSCs or cells derived from the iPSC.
  • the one or more therapeutic agents is a HLA class I proteins.
  • the one or more therapeutic agents is a HLA class II proteins.
  • the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents.
  • the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agent.
  • the second donor polynucleotide template comprises an inducible promoter operably linked to at least one of the one or more therapeutic agent.
  • the pleiopluripotent cell is a pluripotent stem cell.
  • the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
  • the pluripotent stem cell is an induced pluripotent stem cell.
  • the induced pluripotent stem cell is a human induced pluripotent stem cell.
  • this disclosure features a composition comprising a clonal population of any of the pleiopluripotent cells described herein.
  • this disclosure features a pharmaceutical composition comprising a clonal population of pleiopluripotent cells described herein and a pharmaceutically acceptable excipient or carrier.
  • this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site is incorporated site-specifically into the PC-derived cell genome.
  • PC pleiopluripotent cell
  • this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • PC pleiopluripotent cell
  • this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a hematopoietic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site is incorporated site-specifically into the PC-derived cell genome.
  • PC pleiopluripotent cell
  • this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a hematopoietic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • PC pleiopluripotent cell
  • the hematopoietic cell is selected from: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells.
  • HSC hematopoietic stem and progenitor cells
  • MPP hematopoietic multipotent progenitor
  • this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a neuronal cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site integration recognition sites incorporated site-specifically into the PSC-derived cell genome.
  • PC pleiopluripotent cell
  • this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a neuronal cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • PC pleiopluripotent cell
  • the neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron.
  • NPC neural progenitor cell
  • this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a cardiac cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site incorporated site-specifically into the PSC-derived cell genome.
  • this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a cardiac cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • PC pleiopluripotent cell
  • the cardiac cell is selected from: a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte.
  • this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a pancreatic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site incorporated site-specifically into the PC-derived cell genome.
  • PC pleiopluripotent cell
  • this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a pancreatic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • PC pleiopluripotent cell
  • the pancreatic cell is selected from a pancreatic progenitor cell, pancreatic endoderm, a endocrine pancreatic cell, an exocrine pancreatic cell, a islet progenitor, and a beta cell.
  • the PC-derived cell further comprises two integration recognition sites, three integration recognition sites, four integration recognition sites, five integration recognition sites, six integration recognition sites, seven integration recognition sites, eight integration recognition sites, nine integration recognition sites, or ten or more integration recognition sites.
  • At least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a safe harbor locus.
  • at least one of the at least first integration recognition sites is site-specifically incorporated within a locus, disruption of which is capable of reducing allogeneic immune response to the PC-derived cell.
  • at least one of the at least first integration recognition sites is incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
  • the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
  • the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
  • the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA- DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus.
  • the locus is the B2M locus.
  • the locus is the CIITA locus.
  • At least one of the at least first recognition sites is specific for a serine integrase.
  • at least one of the at least first integration recognition sites is an attB or attP site.
  • at least one of the at least first integration recognition sites is a modified attB or attP site.
  • at least one of the at least first integration recognition site is specific for BxB1 or a modified BxB1.
  • At least one of the at least first integration recognition sites is comprised of 38 or 46 nucleotides.
  • the first donor polynucleotide template encodes one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof.
  • inducing at least one of the one or more inducible suicide genes targets the pleiopluripotent cell or a cell matured from the pleiopluripotent cell for cell death.
  • expression of at least one of the one or more inducible suicide gene is driven by a pluripotent stem cell-specific promoter. In some embodiments, expression of the at least one of the one or more inducible suicide genes is driven by a constitutive promoter. In some embodiments, expression of the at least one of the one or more inducible suicide genes is driven by an inducible promoter. In some embodiments, the one or more inducible suicide genes is select from: caspase9, cytosine deaminase, and thymidine kinase. In some embodiments, the one or more inducible suicide genes is a controllable caspase9.
  • the cell further comprises a second inducible suicide gene.
  • the polynucleotide encoding for the second inducible suicide gene is operably linked to the controllable capase9 protein using a 2A self-cleaving peptide or an internal ribosome entry site.
  • the second inducible suicide gene comprises a thymidine kinase.
  • the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells.
  • the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells by inhibiting, blocking, or preventing recognition of the PC-derived cell by macrophages, T- cells, Natural Killer (NK)-cells, antigen-presenting cells, or a combination thereof.
  • the one or more exogenous polypeptides capable of modulating an immune response are capable of serving as a safety switch target.
  • expression of at least one of the one or more exogenous polypeptide capable of modulating an immune response is driven by a pluripotent stem cell-specific promoter.
  • expression of at least one of the one or more exogenous polypeptide capable of modulating an immune response is driven by a constitutive promoter.
  • expression of at least one of the one or more exogenous polypeptide capable of modulating an immune response is driven by an inducible promoter.
  • the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL- 1, B2M-HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof.
  • the donor polynucleotide template encodes a CD47, PDL1, and B2M-HLA-E.
  • the polynucleotide template encoding CD47, PDL1 and B2M-HLA-E are separated by a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof.
  • the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag.
  • the tag is an ALFA tag.
  • the cell further comprises a second integration recognition site site-specifically incorporated into the pleiopluripotent cell genome.
  • the second integration recognition sites is site-specifically incorporated into a safe harbor locus. In some embodiments, the second integration recognition site is site-specifically incorporated within a second locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell. In some embodiments, the second integration recognition site is site-specifically incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
  • the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA- DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus.
  • the locus is the B2M locus.
  • the locus is the CIITA locus.
  • the second integration recognition site is specific for a serine integrase.
  • the second integration recognition sites is different from the at least first integration recognition attB or attP site.
  • the second integration recognition site is an attB or attP site.
  • the second integration ecognition sites is a modified attB or attP site.
  • the second integration ecognition site is specific for BxB1 or a modified BxB1.
  • the second integration recognition sites is comprised of 38 or 46 nucleotides.
  • the donor polynucleotide template encodes one or more orthogonal integration recognition sites.
  • a second donor polynucleotide template is integrated into the PC-derived cell genome at the second integration recognition site site-specifically incorporated into the PC-derived cell genome.
  • the first donor polynucleotide template is encodes one or more therapeutic agents.
  • the one or more therapeutic agents is selected from: transcription factors, receptors, signaling molecules, secreted proteins, cytokines, enzymes, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, and persistence and/or survival of the iPSCs or cells derived from the iPSC.
  • the one or more therapeutic agents is a HLA class I proteins.
  • the one or more therapeutic agents is a HLA class II proteins.
  • the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents.
  • the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agent.
  • the second donor polynucleotide template comprises an inducible promoter operably linked to at least one therapeutic agent.
  • the PC-derived cell or population thereof are human cells.
  • this disclosure features a composition comprising any of the population of PC-derived cells described herein.
  • this disclosure features a pharmaceutical composition comprising any of the population of PC-derived cells described herein and a pharmaceutically acceptable excipient or carrier.
  • this disclosure features a method of treating or ameliorating or preventing a disease or condition in a subject, comprising administering a therapeutically effective amount of any of the PC-derived cells or populations described herein or any of the compositions described herein or any of the pharmaceutical composition described herein.
  • the disease is a cancer.
  • the disease is a muscular and/or the condition is muscle degeneration or muscle injury.
  • the disease is a neuronal disease and/or the condition is neuron degeneration.
  • this disclosure features a method of using a pleiopluripotent cell having at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome, the method comprising: integrating a first donor polynucleotide template into the pleiopluripotent cell genome by introducing into the pleiopluripotent cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the at least one incorporated genomic integration recognition sites by the integrase.
  • the method further comprises selecting the pleiopluripotent cells having the first donor polynucleotide template site- specifically integrated into the genome. [0190] In some embodiments of the method of using the pleiopluripotent cell, the method further comprises expanding the pleiopluripotent cells in a de-differentiated state. [0191] In some embodiments of the method of using the pleiopluripotent cell, the method further comprises cryopreserving the pleiopluripotent cells.
  • the method further comprises directing differentiation of the modified pleiopluripotent cell to a hematopoietic cell.
  • the hematopoietic cell is selected from: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells.
  • HSC hematopoietic stem and progenitor cells
  • MPP hematopoietic multipotent progenitor
  • the method further comprises directing differentiation of the modified pleiopluripotent cell to a neuronal cell.
  • the neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron.
  • NPC neural progenitor cell
  • the method further comprises directing differentiation of the modified pleiopluripotent cell to a cardiac cell.
  • the cardiac cell is selected from: a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte.
  • the method further comprises directing differentiation of the modified pleiopluripotent cell to a pancreatic cell.
  • the cardiac cell is selected from: pancreatic progenitor cell, pancreatic endoderm, an endocrine pancreatic cell, an exocrine pancreatic cell, an islet progenitor, and a beta cell.
  • FIG.1A-1E shows analysis of AttP variants.
  • FIG.1A shows a non-limiting schematic of AttP mutations tested for improving integration efficiency (SEQ ID NOS: 394 and 540-542, respectively, in order of appearance).
  • FIG. 1B shows integration efficiencies of wildtype and mutant AttP sites across a panel of AttB lengths.
  • FIG. 1C shows a non-limiting schematic of multiplexed integration of different cargo sets at specific genomic loci.
  • Three fluorescent cargos (GFP, mCherry, and YFP) are inserted orthogonally at three different loci (ACTB, LMNB1, NOLC1) for in-frame gene tagging.
  • FIG.1D shows orthogonality of top 4 AttB/AttP dinucleotide pairs evaluated for GFP integration with PASTE at the ACTB locus.
  • FIG.2 illustrates a schematic of a non-limiting example of engineered pleiopluripotent cell having a knockout of HLA Class I and/or II, one or more exogenous polypeptides capable of modulating an immune response, one or more inducible suicide genes, one or more adaptors, and optionally one or more beacons for inserting additional therapeutic genes.
  • FIG. 3A illustrates a schematic of a donor polynucleotide template: NANOG- FKBPCasp9-ACTB-mFRBFKBPCasp9-CAG-CD47-PDL1-HLAE-3AttB and integration of the donor polynucleotide into exon 1 of the B2M locus.
  • B2M – beta-2 microglobulin B2M – beta-2 microglobulin.
  • FKBPCasp9 FK506-binding protein fused to caspase 9.
  • ACTB-mFRB-FKBP-Casp9 Actin promoter driving expression of an FRB domain and FKBP-Casp9.
  • CD47 Cluster of Differentiation 47 (i.e., an example exogenous polypeptide capable of modulating an immune response).
  • PD-L1 Programmed death-ligand 1.
  • B2M Beta-2-Microglobulin.
  • HLA-E Major Histocompatibility Complex, Class I, E.
  • FIG. 3B illustrates a schematic of a donor polynucleotide template: PTRE3G-B2M- pA-hPGKprm-Tet3G-pA and integration of the donor polynucleotide template into exon 2 of the CIITA locus. Abbreviations (bottom panel): PTRE3G – TetO promoter. B2M - beta-2 microglobulin.
  • FIG.4 illustrates a schematic of an example inducible suicide gene. Left panel shows non-limiting examples Caspase9-based suicide genes. Right panel shows non-limiting examples of exogenous molecules that control activity of Caspase 9.
  • FIG. 5 illustrates a schematic of single atgRNA and dual atgRNA approaches for beacon placement.
  • FIG.6 shows the source of protospacer sequences for each atgRNA targeting human B2M and human CIITA genes and the dual atgRNA approach for targeting the B2M and CIITA loci.
  • FIGs. 7A-7B illustrates designing of atgRNAs capable of beacon placement into a target genome site. Exemplary 38 or 46 base attB insertion sites are shown.
  • FIG.7A shows a non- limiting example workflow for designing single atgRNA and dual atgRNA approaches for beacon placement.
  • FIG.7B shows a non-limiting example workflow for designing each atgRNA.
  • FIG.8 illustrates a non-limiting example workflow for editing pleiopluripotent cells.
  • FIG.9 illustrates the modified atgRNA sequences for targeting the B2M and CIITA loci.
  • Non-modified RNA are entered as ‘r_’. For example, as rA or rU.2’ O-methyl RNA are entered as ‘m_’.
  • FIGs. 10A-10B illustrate beacon placement in two different iPSC lines (iPSC line 1 was in FIG. 10A and iPSC line 2 was in FIG. 10B) across tested conditions for two different genomic loci in each iPSC line (B2M and CIITA loci). Beacon placement was determined by ddPCR and amp-seq.
  • FIGs.11A-11B illustrate concurrent beacon placement at B2M and CIITA loci using a dual atgRNA approach for beacon placement at each loci. Beacon placement was determined by ddPCR.
  • FIG.11A shows raw data from ddPCR.
  • FIG.11B shows a histogram summarizing data from FIG.11A.
  • FIGs. 12A-12B illustrate beacon placement data for various iPSC lines.
  • FIG. 12A shows raw ddPCR data for beacon placement at the B2M loci and the CIITA loci for 22 iPSC lines.
  • FIG.12B shows a histogram summarizing ddPCR data for three iPSC lines. Lines 1 and 9 show 100% (i.e., bi-allelic) beacon placement at both B2M and CIITA loci.
  • FIG.13 shows Amp-seq data for beacon placement in six iPSC cell lines generated in Example 2.
  • FIGs.14A-14B illustrate a schematic of the digital droplet PCR (ddPCR) assay used to detect integration of a donor polynucleotide at the B2M locus (FIG. 14A) or the CIITA locus (FIG.14B). Similar designs can be used to determine integration at other genomic loci.
  • FIG. 15 shows data for integration of a 6 kilobase (kb) first donor polynucleotide template into the B2M locus (site 1) and a 5kb second donor polynucleotide template into the CIITA locus (site 2).
  • Data is presented as transductions that include a single donor polynucleotide template, thereby measuring a single integration of donor polynucleotide template at either the B2M locus or the CIITA locus (single integration), referred to in FIG.15 as “single.”
  • Data is also presented for transductions that include both the first and second donor polynucleotide templates, thereby showing concurrent integration of the donor polynucleotide templates at the B2M locus and CIITA locus, referred to in FIG.15 as “Duplex”.
  • FIG.16 shows data for programmable gene insertion (i.e., integration) of a 31 kb donor polynucleotide template at the CIITA locus that has an integration recognition site site-specifically incorporated into exon 2.
  • the 31 kb donor polynucleotide (Adv donor) was delivered to the induced pluripotent stem cell (iPSC) using adenovirus at MOIs of 0.01, 0.1, 1, and 10.
  • FIG.17 shows polynucleotide and amino acid sequences for the donor polynucleotide template described in FIG.3A.
  • FIG.17 includes the polynucleotide sequence (SEQ ID NO: 616) of the donor polynucleotide template that is integrated into the B2M locus.
  • FIG.17 also includes the FKBP-linker-Caspase9 amino acid sequence (SEQ ID NO: 662); the mFRB-linker-FKBP- Caspase9-P2A-DeltaTK amino acid sequence (SEQ ID NO: 663); and the alpha tag-CD47-P2A- PDL1-T2A-B2M-HLA-E amino acid sequence (SEQ ID NO: 664).
  • FIG. 662 includes the FKBP-linker-Caspase9 amino acid sequence (SEQ ID NO: 662); the mFRB-linker-FKBP- Caspase9-P2A-DeltaTK amino acid sequence (SEQ ID NO: 663); and the alpha tag-CD47-P2A- PDL1-T2A-B2
  • FIG.18 shows polynucleotide and amino acid sequences for donor polynucleotide template described in FIG.3B.
  • FIG.18 includes the polynucleotide sequence (SEQ ID NO: 617) of the donor polynucleotide template that is integrated into the CIITA locus.
  • FIG.18 also includes the B2M amino acid sequence (SEQ ID NO: 665) and the Tet3G amino acid sequence (SEQ ID NO: 666).
  • FIG.19A shows ddPCR data for percent beacon placement in the CD52 locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis and identified in the key.
  • FIG.19B shows next-generation sequencing data for percent beacon placement in the CD52 locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis that include a subset of the atgRNAs identified in the key in FIG.19A.
  • FIG.20A shows ddPCR data for percent beacon placement in the CISH locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis, which are also described in Example 6.
  • FIG.20B shows next-generation sequencing data for percent beacon placement in the CISH locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis.
  • the pairs in FIG.20B correspond to the pairs in FIG.20A as follows: pair 1 corresponds to SP01-N01, pair 2 corresponds to SP02-N02, pair 5 corresponds to SP05-N04, pair 3 corresponds to SP03-N03, and pair 4 corresponds to SP04-N04.
  • FIG.20A shows next-generation sequencing data for percent beacon placement in the CISH locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis.
  • the pairs in FIG.20B correspond to the pairs in FIG.20A as follows: pair 1 corresponds to SP01-N01, pair 2 corresponds to SP02-N02, pair 5 corresponds to SP05-N04, pair 3 corresponds to SP03-N03, and pair 4 corresponds to SP04-
  • FIG. 21A shows ddPCR data for percent beacon placement in the ADRA2A locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis, which are also described in Example 7.
  • FIG.21B shows next-generation sequencing data for percent beacon placement in the ADRA2A locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis.
  • the pairs in FIG. 21B correspond to the pairs in FIG. 21A as follows: pair 1 corresponds to SP01-N01, pair 2 corresponds to SP02-N02, pair 3 corresponds to SP03-N03, pair 4 corresponds to SP04-N04, and pair 5 corresponds to SP05-N05.
  • FIG. 22 shows ddPCR data for percent beacon placement in the B2M, CIITA, and CD52 loci in a wild type iPSC line following electroporation with the indicated combinations of atgRNA pairs.
  • FIG.23 shows ddPCR data for percent beacon placement in the TRAC and AAVS1 loci in iPSC clone # 17 following electroporation with the atgRNA pairs indicated on the x-axis.
  • FIG.24 shows a non-limiting example of the clonal isolation process for generating an iPSC clone. The overall process takes roughly a month from electroporation to NGS analysis. First, electroporation (EP) is used to place beacons in the iPS cells.
  • EP electroporation
  • Electroporated iPSCs are grown for 72 hours and part of the iPSC culture is harvested for genomic DNA to get an initial read of beacon placement. If cells are positive for beacon placement, electroporation is scaled up from a 48 well plate to a 24 well plate and then eventually to 6 wells. A confluent 6 well population of cells is diluted such that a single cell is placed in each well of a 96-well plate across multiple 96 well plates. These cells take roughly two weeks to reach the point of needing to be harvested. Cell growth is monitored and only clones with good morphology and growth rates are expanded and/or harvested for a second round of ddPCR.
  • FIG. 26 shows ddPCR data for programmable gene insertion of four different donor polynucleotide templates at the B2M, CIITA, TRAC, and AAVS1 loci in iPSC clones #16 and #29.
  • Each donor polynucleotide template includes an AttP site having a different central dinucleotide that enables integration at its corresponding (cognate) AttB site that has been site-specifically integrated into the iPS cell genome at one of the indicated loci.
  • Controls include no BxB1 control (i.e., donor polynucleotide templates but no BxB1) and a no donor polynucleotide template control (i.e., a BxB1 polypeptide or polynucleotide encoding a BxB1 polypeptide but no donor polynucleotide templates.
  • FIG. 27 shows ddPCR data for programmable gene insertion of four different donor polynucleotide templates at the B2M, CIITA, TRAC, and AAVS1 loci in iPSC clones #30.
  • Each donor polynucleotide template includes an AttP site having a different central dinucleotide that enables integration at its corresponding (cognate) AttB site that has been site-specifically integrated into the iPS cell genome at one of the indicated loci. 6.
  • the methods described herein enable multiplexed programmable gene insertion whereby multiple edits (e.g., beacon placement, donor polynucleotide incorporation, or a combination thereof) can be performed sequentially or simultaneously in a single pleiopluripotent cell, thereby reducing the time and cost associated with conventional methods of editing pleiopluripotent cells. Additionally, the methods described herein avoid the need for inducing double strand breaks, which increases accuracy of genome integration and reduces likelihood of off-target events.
  • multiple edits e.g., beacon placement, donor polynucleotide incorporation, or a combination thereof
  • a beacon e.g., an integration recognition site
  • a particular genomic location to guide integration of the donor polynucleotide template increases integration efficiency of the donor polynucleotide template.
  • the methods described herein enable integration of larger donor polynucleotide template sequences compared to conventional methods which have payload capacity limitations. This is enabled, at least in part, by using single atgRNA (and optionally a nicking gRNA) or dual atgRNA to guide precise beacon placement (i.e., incorporation of one or more integration recognition sites) at desired loci.
  • pleiopluripotent cells that include one or more of (i) knockout of HLA Class I and/or Class II molecules, (ii) expression of self-molecules (e.g., one or more exogenous polypeptides capable of modulating an immune response), (iii) expression one or more controllable (e.g., inducible) suicide genes, (iv) expression of one or more adaptors (e.g., a tag), and (v) one or more landing pads (e.g., beacons) incorporated into the genome for integrating additional therapeutic agents (see FIG.2).
  • self-molecules e.g., one or more exogenous polypeptides capable of modulating an immune response
  • controllable e.g., inducible
  • suicide genes e.g., a controllable suicide genes
  • adaptors e.g., a tag
  • landing pads e.g., beacons
  • Cells comprising one or more of these features provide a source of cells suitable for differentiation to a cell type of interest (e.g., without limitation, a hematopoietic cell, a neuronal cell, a cardiac cell, and a pancreatic cell) and use in cell therapy (e.g., allogeneic cell therapy).
  • a cell type of interest e.g., without limitation, a hematopoietic cell, a neuronal cell, a cardiac cell, and a pancreatic cell
  • cell therapy e.g., allogeneic cell therapy.
  • the terms “gene editor polynucleotide” refers to a polynucleotide sequence encoding the gene editor polypeptide.
  • Such an enzyme or enzyme fusion may contain DNA or RNA targetable nuclease protein (i.e., Cas protein, ADAR, or ADAT), wherein target specificity is mediated by a complexed nucleic acid (i.e., guide RNA).
  • RNA targetable nuclease protein i.e., Cas protein, ADAR, or ADAT
  • target specificity is mediated by a complexed nucleic acid (i.e., guide RNA).
  • Such an enzyme or enzyme fusion may be a DNA/RNA targetable protein, wherein target specificity is mediated by internal, conjugated, fused, or linked amino acids, such as within TALENs, ZFNs, or meganucleases.
  • a gene editor can demonstrate targeted nuclease activity, targeted binding with no nuclease activity, or targeted nickase activity (or cleavase activity).
  • a gene editor comprising a targetable protein may be fused or linked to one or more proteins or protein fragment motifs. Gene editors may be fused, linked, complexed, operate in cis or trans to one or more integrase, recombinase, polymerase, telomerase, reverse transcriptase, or invertase.
  • a gene editor can be a prime editor fusion protein or a gene writer fusion protein.
  • Prime editor fusion protein describes a protein that is used in prime editing.
  • Prime editing uses CRISPR enzyme that nicks or cuts only single strand of double stranded DNA, i.e., a nickase; the nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts.
  • a nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts.
  • Such an enzyme can be a catalytically impaired Cas9 endonuclease (a nickase).
  • a nickase Such an enzyme can be a Casl2a/b, MAD7, or variant thereof.
  • the nickase is fused to an engineered reverse transcriptase (RT).
  • the nickase is programmed (directed) with an attachment site-containing guide RNA (or a prime-editing guide RNA (pegRNA)).
  • an attachment site-containing guide RNA or a prime-editing guide RNA (pegRNA)
  • pegRNA prime-editing guide RNA
  • atgRNA attachment site-containing guide RNA
  • the nickase may be programmed (directed) with an atgRNA.
  • the nickase is a catalytically-impaired Cas9 endonuclease, a Cas9 nickase, that is fused to the reverse transcriptase.
  • the Cas9 nickase part of the protein is guided to the DNA target site by the atgRNA (or pegRNA), whereby a nick or single stranded cut occurs.
  • the reverse transcriptase domain then uses the atgRNA (or pegRNA) to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand.
  • the edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand.
  • the prime editor (PE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA).
  • cleavase e.g., cleavase I enzyme
  • an additional agent or agents may be added that improve the efficiency and outcome purity of the prime edit.
  • the agent may be chemical or biological and disrupt DNA mismatch repair (MMR) processes at or near the edit site (i.e., PE4 and PE5 and PEmax architecture by Chen et al. Cell, 184, 1-18, October 28, 2021; Chen et al. is incorporated herein by reference).
  • MMR DNA mismatch repair
  • the agent is a MMR-inhibiting protein.
  • the MMR-inhibiting protein is dominant negative MMR protein.
  • the dominant negative MMR protein is MLH1dn.
  • the MMR-inhibiting agent is incorporated into the single nucleic acid construct design described herein.
  • the MMR-inhibiting agent is linked or fused to the prime editor protein fusion, which may or may not have a linked or fused integrase.
  • the MMR-inhibiting agent is linked or fused to the Gene WriterTM protein, which may or may not have a linked or fused integrase.
  • the prime editor or gene editor system can be used to achieve DNA deletion and replacement.
  • the DNA deletion replacement is induced using a pair of atgRNAs (or pegRNA) that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair (i.e., PrimeDel by Choi et al. Nat. Biotechnology, October 14, 2021; Choi et al. is incorporated herein by reference and TwinPE by Anzalone et al., Nat. Biotech. 40: 731–740 (2022); Anzalone et al. is incorporated herein by reference).
  • the DNA deletion is induced using a single atgRNA.
  • the DNA deletion and replacement is induced using a wild type Cas9 prime editor (PE-Cas9) system (i.e., PEDAR by Jiang et al. Nat. Biotechnology, October 14, 2021; Jiang et al. is incorporated herein by reference).
  • the DNA replacement is an integrase target recognition site or recombinase target recognition site.
  • the constructs and methods described herein may be utilized to incorporate the pair of pegRNAs used in PrimeDel, TwinPE (WO2021226558, which is hereby incorporated by reference herein in its entirety), or PEDAR, the prime editor fusion protein or Gene Writer protein, optionally a nickase guide RNA (ngRNA), an integrase, a nucleic acid cargo, and optionally a recombinase into a single nucleic acid construct described herein.
  • the integrase may be directly linked, for example by a peptide linker, to the prime editor fusion or gene writer protein.
  • the prime editors can refer to a retrovirus or lentivirus reverse transcriptase such as a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (RT) fused to a CRISPR enzyme nickase such as a Cas9 H840A nickase, a Cas9nickase.
  • the prime editors can refer to a retrovirus or lentivirus reverse transcriptase such as a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (RT) fused to a cleavase.
  • the RT can be fused at, near or to the C-terminus of a Cas9nickase, e.g., Cas9 H840A. Fusing the RT to the C-terminus region, e.g., to the C-terminus, of the Cas9 nickase may result in higher editing efficiency.
  • a complex is called PEI.
  • the CRISPR enzyme nickase e.g., Cas9(H840A), i.e., a Cas9nickase
  • the CRISPR enzyme nickase instead of being a Cas9 (H840A), i.e., instead of being a Cas9 nickase, the CRISPR enzyme nickase instead can be a CRISPR enzyme that naturally is a nickase or cuts a single strand of double stranded DNA; for instance, the CRISPR enzyme nickase can be Casl2a/b. Alternatively, the CRISPR enzyme nickase can be another mutation of Cas9, such as Cas9(Dl0A).
  • a CRISPR enzyme such as a CRISPR enzyme nickase, such as Cas9 (wild type), Cas9(H840A), Cas9(Dl0A) or Cas 12a/b nickase can be fused in some embodiments to a pentamutant of M-MLV RT (D200N/ L603W/ T330P/ T306K/ W313F), whereby there can be up to about 45-fold higher efficiency, and this is called PE2.
  • a CRISPR enzyme nickase such as Cas9 (wild type), Cas9(H840A), Cas9(Dl0A) or Cas 12a/b nickase
  • a pentamutant of M-MLV RT D200N/ L603W/ T330P/ T306K/ W313F
  • the M-MLV RT comprise one or more of the mutations Y8H, P51L, S56A, S67R, E69K, Vl29P, L139P, Tl97A, H204R, V223H, T246E, N249D, E286R, Q2911, E302K, E302R, F309N, M320L, P330E, L435G, L435R, N454K, D524A, D524G, D524N, E562Q, D583N, H594Q, E607K, D653N, and L671P. Specific M-MLV RT mutations are shown in Table 1.
  • the reverse transcriptase can also be a wild-type or modified transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV RT), Feline Immunodeficiency Virus reverse transcriptase (FIV-RT), FeLV-RT (Feline leukemia virus reverse transcriptase), HIV-RT (Human Immunodeficiency Virus reverse transcriptase).
  • RTX transcription xenopolymerase
  • AMV RT avian myeloblastosis virus reverse transcriptase
  • FV-RT Feline Immunodeficiency Virus reverse transcriptase
  • FeLV-RT FeLV-RT
  • Feline leukemia virus reverse transcriptase FeLV-RT
  • HIV-RT Human Immunodeficiency Virus reverse transcriptase
  • the reverse transcriptase can be a fusion of MMuLV to the Sto7d DNA binding domain (see Ionnidi et al.;
  • PE3, PE3b, PE4, PE5, and/or PEmax which a skilled person can incorporate into the gene editor polypeptide, involves nicking the non-edited strand, potentially causing the cell to remake that strand using the edited strand as the template to induce HR.
  • the nicking of the non-edited strand can involve the use of a nicking guide RNA (ngRNA).
  • ngRNA nicking guide RNA
  • the skilled person can readily incorporate into a gene editor polypeptide described herein a prime editing or CRISPR system.
  • Prime editors can be found in the following: WO2020/191153, WO2020/191171, WO2020/191233, WO2020/191234, WO2020/191239, WO2020/191241, WO2020/191242, WO2020/191243, WO2020/191245, WO2020/191246, WO2020/191248, WO2020/191249, each of which is incorporated by reference herein in its entirety.
  • the skilled person can incorporate the selected CRISPR enzyme, as part of the gene editor composition described herein.
  • Additional gene editor polypeptides are as described in WO 2023/076898; WO 2023/015014; WO 2023/070062; WO2023288332; WO2023015318; WO2023004439; WO2023070110; WO2022256714; and WO2023283092, each of which are hereby incorporated by reference in their entireties.
  • Additional gene editor polypeptides are as described in U.S. Patent Pub.2023/0059368, which is hereby incorporated by reference in its entirety.
  • the prime editor protein Prior to RT-mediated edit incorporation, the prime editor protein (1) site- specifically targets a genomic locus and (2) performs a catalytic cut or nick. These steps are typically performed by a CRISPR-Cas.
  • the Cas protein may be substituted by other nucleic acid programmable DNA binding proteins (napDNAbp) such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or meganucleases.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • meganucleases meganucleases
  • a Gene Writer protein comprises: (A) a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain, and either (x) an endonuclease domain that contains DNA binding functionality or (y) an endonuclease domain and separate DNA binding domain; and (B) a template RNA comprising (i) a sequence that binds the polypeptide and (ii) a heterologous insert sequence.
  • the prime editor or Gene Writer protein fusion or prime editor protein linked or fused to an integrase is expressed as a split construct.
  • the split construct in reconstituted in a cell.
  • the split construct can be fused or ligated via intein protein splicing.
  • the split construct can be reconstituted via protein-protein inter-molecular bonding and/or interactions.
  • the split construct can be reconstituted via chemical, biological, or environmental induced oligomerization.
  • the split construct can be reconstituted via nanobody binding ALFA-tagged proteins.
  • the split construct can be adapted into one or more single nucleic acid polynucleotides.
  • an integrase or recombinase is directly linked or fused, for example by a peptide linker, which may be cleavable or non-cleavable, to the prime editor fusion protein (i.e., fused Cas9 nickase-reverse transcriptase) or Gene Writer protein.
  • Suitable linkers for example between the Cas9, RT, and integrase, may be selected from Table 3: Table 3 6.2.
  • Type II CRISPR proteins [0247] The skilled person can incorporate a selected CRISPR enzyme, described below, as part of the prime editor fusion, as a component of the gene editor polypeptide described herein.
  • Streptococcus pyogenes Cas9 (SpCas9), the most common enzyme used in genome-editing applications, is a large nuclease of 1368 amino acid residues. Advantages of SpCas9 include its short, 5′-NGG-3′ PAM and very high average editing efficiency.
  • SpCas9 consists of two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe.
  • the REC lobe can be divided into three regions, a long a helix referred to as the bridge helix (residues 60–93), the REC1 (residues 94–179 and 308–713) domain, and the REC2 (residues 180–307) domain.
  • the NUC lobe consists of the RuvC (residues 1–59, 718–769, and 909–1098), HNH (residues 775–908), and PAM-interacting (PI) (residues 1099–1368) domains.
  • the negatively charged sgRNA:target DNA heteroduplex is accommodated in a positively charged groove at the interface between the REC and NUC lobes.
  • the RuvC domain is assembled from the three split RuvC motifs (RuvC I–III) and interfaces with the PI domain to form a positively charged surface that interacts with the 30 tail of the sgRNA.
  • the HNH domain lies between the RuvC II–III motifs and forms only a few contacts with the rest of the protein. Structural aspects of SpCas9 are described by Nishimasu et al., Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA, Cell 156, 935-949, February 27, 2014.
  • REC lobe The REC lobe includes the REC1 and REC2 domains.
  • the REC2 domain does not contact the bound guide:target heteroduplex, indicating that truncation of REC lobe may be tolerated by SpCas9.
  • SpCas9 mutant lacking the REC2 domain (D175–307) retained ⁇ 50% of the wild-type Cas9 activity, indicating that the REC2 domain is not critical for DNA cleavage.
  • PAM-Interacting domain The NUC lobe contains the PAM-interacting (PI) domain that is positioned to recognize the PAM sequence on the noncomplementary DNA strand.
  • RuvC domain The RuvC nucleases of SpCas9 have an RNase H fold and four catalytic residues, Asp10 (Ala), Glu762, His983, and Asp986, that are critical for the two-metal cleavage of the noncomplementary strand of the target DNA.
  • the Cas9 RuvC domain has other structural elements involved in interactions with the guide:target heteroduplex (an end-capping loop between ⁇ 42 and ⁇ 43) and the PI domain/stem loop 3 ( ⁇ hairpin formed by ⁇ 3 and ⁇ 4).
  • HNH domain SpCas9 HNH nucleases have three catalytic residues, Asp839, His840, and Asn863 and cleave the complementary strand of the target DNA through a single-metal mechanism.
  • sgRNA DNA recognition: The sgRNA guide region is primarily recognized by the REC lobe.
  • the backbone phosphate groups of the guide region interact with the REC1 domain (Arg165, Gly166, Arg403, Asn407, Lys510, Tyr515, and Arg661) and the bridge helix (Arg63, Arg66, Arg70, Arg71, Arg74, and Arg78).
  • the 20-hydroxyl groups of G1, C15, U16, and G19 hydrogen bond with Val1009, Tyr450, Arg447/Ile448, and Thr404, respectively.
  • RNA-guided DNA targeting: SpCas9 recognizes the guide:target heteroduplex in a sequence-independent manner.
  • the backbone phosphate groups of the target DNA interact with the REC1 (Asn497, Trp659, Arg661, and Gln695), RuvC (Gln926), and PI (Glu1108) domains.
  • the C2’ atoms of the target DNA form van der Waals interactions with the REC1 domain (Leu169, Tyr450, Met495, Met694, and His698) and the RuvC domain (Ala728).
  • the terminal base pair of the guide:target heteroduplex (G1:C20’) is recognized by the RuvC domain via end-capping interactions; the sgRNA G1 and target DNA C20’ nucleobases interact with the Tyr1013 and Val1015 side chains, respectively, whereas the 20-hydroxyl and phosphate groups of sgRNA G1 interact with Val1009 and Gln926, respectively.
  • the nucleobase of the flipped U44 is sandwiched between Tyr325 and His328, with its N3 atom hydrogen bonded with Tyr325, whereas the nucleobase of the unpaired G43 stacks with Tyr359 and hydrogen bonds with Asp364.
  • the nucleobases of G21 and U50 in the G21:U50 wobble pair stack with the terminal C20:G10 pair in the guide:target heteroduplex and Tyr72 on the bridge helix, respectively, with the U50 O4 atom hydrogen bonded with Arg75.
  • A51 adopts the syn conformation and is oriented in the direction opposite to U50.
  • Stem loop 1 is primarily recognized by the REC lobe, together with the PI domain.
  • the backbone phosphate groups of stem loop 1 interact with the REC1 domain (Leu455, Ser460, Arg467, Thr472, and Ile473), the PI domain (Lys1123 and Lys1124), and the bridge helix (Arg70 and Arg74), with the 20-hydroxyl group of G58 hydrogen bonded with Leu455.
  • A52 interacts with Phe1105 through a face-to-edge p-p stacking interaction, and the flipped U59 nucleobase hydrogen bonds with Asn77.
  • the single-stranded linker and stem loops 2 and 3 are primarily recognized by the NUC lobe.
  • the backbone phosphate groups of the linker (nucleotides 63–65 and 67) interact with the RuvC domain (Glu57, Lys742, and Lys1097), the PI domain (Thr1102), and the bridge helix (Arg69), with the 20-hydroxyl groups of U64 and A65 hydrogen bonded with Glu57 and His721, respectively.
  • the C67 nucleobase forms two hydrogen bonds with Val1100.
  • Stem loop 2 is recognized by Cas9 via the interactions between the NUC lobe and the non-Watson-Crick A68:G81 pair, which is formed by direct (between the A68 N6 and G81 O6 atoms) and water-mediated (between the A68 N1 and G81 N1 atoms) hydrogen-bonding interactions.
  • the A68 and G81 nucleobases contact Ser1351 and Tyr1356, respectively, whereas the A68:G81 pair interacts with Thr1358 via a water-mediated hydrogen bond.
  • Stem loop 3 interacts with the NUC lobe more extensively, as compared to stem loop 2.
  • the backbone phosphate group of G92 interacts with the RuvC domain (Arg40 and Lys44), whereas the G89 and U90 nucleobases hydrogen bond with Gln1272 and Glu1225/Ala1227, respectively.
  • the A88 and C91 nucleobases are recognized by Asn46 via multiple hydrogen- bonding interactions.
  • Cas9 proteins smaller than SpCas9 allow more efficient packaging of nucleic acids encoding CRISPR systems, e.g., Cas9 and sgRNA into one rAAV (“all-in-one-AAV”) particle.
  • Small Cas9 proteins can be advantageous for multidomain-Cas-nuclease-based systems for prime editing.
  • Well characterized smaller Cas9 proteins include Staphylococcus aureus (SauCas9, 1053 amino acid residues) and Campylobacter jejuni (CjCas9, 984 amino residues).
  • Staphylococcus lugdunensis (Slu) Cas9 as having genome-editing activity and provided homology mapping to SpCas9 and SauCas9 to facilitate generation of nickases and inactive (“dead”) enzymes (Schmidt et al., 2021, Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases. Nat Commun 12, 4219. doi.org/10.1038/s41467-021- 24454-5) and engineered nucleases with higher cleavage activity by fragmenting and shuffling Cas9 DNAs.
  • the small Cas9s and nickases are useful in the instant disclosure.
  • the Cas9 proteins used herein may also include other “Cas9 variants” having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art.
  • Cas9 variants having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild
  • a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9.
  • the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9.
  • a reference Cas9 e.g., a gRNA binding domain or a DNA-cleavage domain
  • the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.
  • the disclosure also may utilize Cas9 fragments that retain their functionality and that are fragments of any herein disclosed Cas9 protein.
  • the Cas9 fragment is at least 100 amino acids in length.
  • the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.
  • the prime editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 variants. 6.2.1. Table 4. Cas9 Orthologs.
  • prime editors utilized herein comprise CRISPR-Cas system enzymes other than type II enzymes.
  • prime editors comprise type V or type VI CRISPR-Cas system enzymes. It will be appreciated that certain CRISPR enzymes exhibit promiscuous ssDNA cleavage activity and appropriate precautions should be considered.
  • prime editors comprise a nickase or a dead CRISPR with nuclease function comprised in a different component.
  • the nucleic acid programmable DNA binding proteins utilized herein include, without limitation, Cas9 (e.g., dCas9 and nCas9), Cas12a (Cpf1), Cas12b1 (C2c1), Cas12b2, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), C2c4, C2c5, C2c8, C2c9, C2c10, Cas13a (C2c2), Cas13b (C2c6), Cas13c (C2c7), Cas13d, and Argonaute.
  • Cas9 e.g., dCas9 and nCas9
  • Cas9 e.g., dCas9 and nCas9
  • Cas9 e.g., dCas9 and nCas9
  • Cas9 e.g., dCas9 and nC
  • Cas-equivalents further include those described in Makarova et al., “C2c2 is a single-component programmable RNA- guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) and Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol.1. No.5, 2018, the contents of which are incorporated herein by reference.
  • One example of a nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (i.e, Cas12a (Cpf1)).
  • Cas12a (Cpf1) is also a Class 2 CRISPR effector, but it is a member of type V subgroup of enzymes, rather than the type II subgroup. It has been shown that Cas12a (Cpf1) mediates robust DNA interference with features distinct from Cas9.
  • Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T- rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break.
  • Cpf1-family proteins Two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells.
  • Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p.949-962; the entire contents of which is hereby incorporated by reference. 6.3.
  • Type V CRISPR proteins [0267]
  • prime editors used herein comprise the type V CRISPR family includes Francisella novicida U112 Cpf1 (FnCpf1) also known as FnCas12a.
  • FnCpf1 adopts a bilobed architecture with the two lobes connected by the wedge (WED) domain.
  • the N-terminal REC lobe consists of two a-helical domains (REC1 and REC2) that have been shown to coordinate the crRNA-target DNA heteroduplex.
  • the C-terminal NUC lobe consists of the C-terminal RuvC and Nuc domains involved in target cleavage, the arginine-rich bridge helix (BH), and the PAM- interacting (PI) domain.
  • the repeat-derived segment of the crRNA forms a pseudoknot stabilized by intra-molecular base-pairing and hydrogen-bonding interactions.
  • the pseudoknot is coordinated by residues from the WED, RuvC, and REC2 domains, as well as by two hydrated magnesium cations.
  • nucleotides 1–5 of the crRNA are ordered in the central cavity of FnCas12a and adopt an A-form-like helical conformation. Conformational ordering of the seed sequence is facilitated by multiple interactions between the ribose and phosphate moieties of the crRNA backbone and FnCpf1 residues in the WED and REC1 domains. These include residues Thr16, Lys595, His804, and His881 from the WED domain and residues Tyr47, Lys51, Phe182, and Arg186 from the REC1 domain.
  • FnCas12a-crRNA complex further reveals that the bases of the seed sequence are solvent exposed and poised for hybridization with target DNA.
  • Structural aspects of FnCpf1 are described by Swarts et al., Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a, Molecular Cell 66, 221-233, April 20, 2017. [0268]
  • Pre-crRNA processing Essential residues for crRNA processing include His843, Lys852, and Lys869.
  • R-loop formation The crRNA-target DNA strand heteroduplex is enclosed in the central cavity formed by the REC and NUC lobes and interacts extensively with the REC1 and REC2 domains.
  • the PAM-containing DNA duplex comprises target strand nucleotides dT0–dT8 and non-target strand nucleotides dA(8)*–dA0* and is contacted by the PI, WED, and REC1 domains.
  • the 5’-TTN-3’ PAM is recognized in FnCas12a by a mechanism combining the shape- specific recognition of a narrowed minor groove, with base-specific recognition of the PAM bases by two invariant residues, Lys671 and Lys613.
  • the duplex of the target DNA is disrupted by the side chain of residue Lys667, which is inserted between the DNA strands and forms a cation- ⁇ stacking interaction with the dA0–dT0* base pair.
  • the phosphate group linking target strand residues dT(-1) and dT0 is coordinated by hydrogen-bonding interactions with the side chain of Lys823 and the backbone amide of Gly826.
  • Target strand residue dT(-1) bends away from residue T0, allowing the target strand to interact with the seed sequence of the crRNA.
  • the non-target strand nucleotides dT1*–dT5* interact with the Arg692- Ser702 loop in FnCas12a through hydrogen-bonding and ionic interactions between backbone phosphate groups and side chains of Arg692, Asn700, Ser702, and Gln704, as well as main-chain amide groups of Lys699, Asn700, and Ser702.
  • Alanine substitution of Q704 or replacement of residues Thr698–Ser702 in FnCas12a with the sequence Ala-Gly3 (SEQ ID NO: 115) substantially reduced DNA cleavage activity, suggesting that these residues contribute to R-loop formation by stabilizing the displaced conformation of the nontarget DNA strand.
  • the crRNA-target strand heteroduplex is terminated by a stacking interaction with a conserved aromatic residue (Tyr410). This prevents base pairing between the crRNA and the target strand beyond nucleotides U20 and dA(-20), respectively. Beyond this point, the target DNA strand nucleotides re-engage the non-target DNA strand, forming a PAM-distal DNA duplex comprising nucleotides dC(-21)–dA(-27) and dG21*–dT27*, respectively. The duplex is confined between the REC2 and Nuc domains at the end of the central channel formed by the REC and NUC lobes.
  • Target DNA cleavage FnCpf1 can independently accommodate both the target and non-target DNA strands in the catalytic pocket of the RuvC domain.
  • the RuvC active site contains three catalytic residues (D917, E1006, and D1255). Structural observations suggest that both the target and non-target DNA strands are cleaved by the same catalytic mechanism in a single active site in Cpf1/Cas12a enzymes.
  • nuclease comprises a Cas12f effector.
  • Small CRISPR- associated effector proteins belonging to the type V-F subtype have been identified through the mining of sequence databases and members classified into Cas12f1 (Cas14a and type V-U3), Cas12f2 (Cas14b) and Cas12f3 (Cas14c, type V-U2 and U4).
  • Exemplary CRISPR-Cas proteins and enzymes used in the Prime Editors herein include the following without limitation. 6.3.1. Table 5. Cas12a orthologs 6.3.2. Table 6. Cas12b (C2c1) Orthologs Table 6 - Cas12b (C2c1) orthologs 6.3.3. Table 7. Cas12c (C2c3) Orthologs
  • Protospacer Adjacent Motif refers to an approximately 2-6 base pair DNA sequence (or a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-long nucleotide sequence) that is an important targeting component of a Cas9 nuclease.
  • the PAM sequence is on either strand, and is downstream in the 5' to 3' direction of Cas9 cut site.
  • the canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5'-NGG-3' wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases.
  • N is any nucleobase followed by two guanine (“G”) nucleobases.
  • G guanine
  • Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms.
  • any given Cas9 nuclease may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence.
  • the PAM specificity can be modified by introducing one or more mutations, including (a) D1135V, R1335Q, and T1337R “the VQR variant”, which alters the PAM specificity to NGAN or NGNG, (b) D1135E, R1335Q, and T1337R “the EQR variant”, which alters the PAM specificity to NGAG, and (c) D1135V, G1218R, R1335E, and T1337R “the VRER variant”, which alters the PAM specificity to NGCG.
  • Cas9 enzymes from different bacterial species can have varying PAM specificities and in some embodiments are therefore chosen based on the desired PAM recognition.
  • Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN.
  • Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT.
  • Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW.
  • Cas9 from Treponema denticola (TdCas) recognizes NAAAAC.
  • TdCas Treponema denticola
  • non- SpCas9s bind a variety of PAM sequences, which makes them useful to expand the range of sequences that can be targeted according to the invention.
  • non-SpCas9s may have other characteristics that make them more useful than SpCas9.
  • Cas9 from Staphylococcus aureus is about 1 kilobase smaller than SpCas9, so it can be packaged into adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • Prime editing uses CRISPR enzyme that nicks or cuts only single strand of double stranded DNA, i.e., a nickase; and a nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts.
  • a nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts.
  • Such an enzyme can be a catalytically-impaired Cas9 endonuclease (a nickase).
  • a nickase Such an enzyme can be a Casl2a/b, MAD7, or variant thereof.
  • the nickase is fused to an engineered reverse transcriptase (RT).
  • the nickase is programmed (directed) with a attachment site-containing guide RNA.
  • the atgRNA both specifies the target site and encodes the desired edit.
  • the nickase is a catalytically-impaired Cas9 endonuclease, a Cas9 nickase, that is fused to the reverse transcriptase.
  • the Cas9 nickase part of the protein is guided to the DNA target site by the pegRNA, whereby a nick or single stranded cut occurs.
  • the reverse transcriptase domain then uses the pegRNA to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand.
  • PE1 refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a wild type MMLV RT having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]- [linker]-[MMLV_RT(wt)] + a desired atgRNA.
  • the prime editors disclosed herein is comprised of PE1.
  • PE2 refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]- [linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] + a desired PEgRNA.
  • the prime editors disclosed herein is comprised of PE2.
  • the prime editors disclosed herein is comprised of PE2 and co-expression of MMR protein MLH1dn, that is PE4.
  • PE3 refers to PE2 plus a second-strand nicking guide RNA that complexes with the PE2 and introduces a nick in the non-edited DNA strand. The induction of the second nick increases the chances of the unedited strand, rather than the edited strand, to be repaired.
  • the prime editors disclosed herein is comprised of PE3.
  • the prime editors disclosed herein is comprised of PE3 and co-expression of MMR protein MLH1dn, that is PE5.
  • PE3b refers to PE3 but wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence with mismatches to the unedited original allele that matches only the edited strand. Using this strategy, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place. 6.6.
  • a prime editing complex consists of a type II CRISPR PE protein containing an RNA-guided DNA-nicking domain fused to a reverse transcriptase (RT) domain and complexed with a pegRNA.
  • the pegRNA comprises (5’ to 3’) a spacer that is complementary to the target sequence of a genomic DNA, a nickase (e.g. Cas9) binding site, a reverse transcriptase template including editing positions, and primer binding site (PBS).
  • the PE–pegRNA complex binds the target DNA and the CRISPR protein nicks the PAM- containing strand.
  • the resulting 3′ end of the nicked target hybridizes to the primer-binding site (PBS) of the pegRNA, then primes reverse transcription of new DNA containing the desired edit using the RT template of the pegRNA.
  • PBS primer-binding site
  • the overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template/primer binding site appended to the 3’ end. The structure leaves the PBS at the 3’ end of the pegRNA free to bind to the nicked strand complementary to the target which forms the primer for reverse transcription.
  • Guide RNAs of CRISPRs differ in overall structure.
  • the spacer of a type II gRNA is located at the 5’ end
  • the spacer of a type V gRNA is located towards the 3’ end, with the CRISPR protein (e.g. Cas12a) binding region located toward the 5’ end.
  • the regions of a type V pegRNA are rearranged compared to a type II pegRNA.
  • the overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template/primer binding site appended to the 3’ end.
  • the pegRNA comprises (5’ to 3’) a CRISPR protein-binding region, a spacer which is complementary to the target sequence of a genomic DNA, a reverse transcriptase template including editing positions, and primer binding site (PBS).
  • the guide RNA or guide RNA complex is capable of binding a DNA binding nickase selected from the group consisting of: Cas9-D10A, Cas9-H840A, Cas12a/b/c/d/e nickase, CasX nickase, SaCas9 nickase, and CasY nickase.
  • the nickase is linked or fused to one or more of a reverse transcriptase.
  • the nickase is linked or fused to one or more of a reverse transcriptase and integrase. In certain embodiments, the nickase is linked or fused to one or more of an integrase. 6.7.
  • Attachment Site-Containing Guide RNA [0289]
  • the term "attachment site-containing guide RNA” (atgRNA) and the like refer to an extended single guide RNA (sgRNA) comprising a primer binding site (PBS), a reverse transcriptase (RT) template sequence, and wherein the RT template encodes for an integration recognition site or a recombinase recognition site that can be recognized by a recombinase, integrase, or transposase.
  • the RT template comprises a clamp sequence and an integration recognition site.
  • an atgRNA may be referred to as a guide RNA.
  • an integration recognition site or recombinase target recognition site incorporated into the pegRNA is referred to as an attachment site containing guide RNA (atgRNA).
  • atgRNA guide RNA
  • the term “cognate integration recognition site” or “integration cognate” or “cognate pair” refers to a first integration recognition site (e.g., any of the integration recognition sites described herein) and a second integration recognition site (e.g., any of the integration recognition sites described herein) that can be recombined.
  • Recombination between a first integration recognition site (e.g., any of the integration recognition sites described herein) and a second recognition site (e.g., any of the integration recognition sites described herein) is mediated by functional symmetry between the two integration recognition sites and the central dinucleotide of each of the two integration recognition sites.
  • a first integration recognition site e.g., any of the integration recognition sites described herein
  • a second integration recognition site e.g., any of the integration recognition sites described herein
  • a non-limiting example of a cognate pair include an attB site and an attP site, whereby a serine integrase mediates recombination between the attB site and the attP site.
  • FIGs.1A-1E show optimization of the integration recognition site.
  • an atgRNA comprises a reverse transcriptase template that encodes, partially or in its entirety, an integration recognition site (also referred to as an integration target recognition site) or a recombinase recognition site (also referred to as a recombinase target recognition site).
  • the integration target recognition site which is to be placed at a desired location in the genome or intracellular nucleic acid, is referred to as a “beacon,” a “beacon” site or an “attachment site” or an “integration recognition site” or a “landing pad” or “landing site.”
  • An integration target recognition site or recombinase target recognition site incorporated into the pegRNA is referred to as an attachment site containing guide RNA (atgRNA).
  • an atgRNA comprises a reverse transcriptase template that encodes, partially or in entirety, for an integration target recognition site or recombinase target recognition site.
  • the integration target recognition site which is to be place at a desired location in the genome, is referred to as a “beacon site” or an “attachment site” or a “landing pad” or “landing site.”
  • the primer binding site allows the 3' end of the nicked DNA strand to hybridize to the atgRNA, while the RT template serves as a template for the synthesis of edited genetic information.
  • the atgRNA is capable for instance, without limitation, of (i) identifying the target nucleotide sequence to be edited and (ii) encoding new genetic information that replaces (or in some cases adds) the targeted sequence.
  • the atgRNA is capable of (i) identifying the target nucleotide sequence to be edited and (ii) encoding an integration site that replaces (or inserts/deletes within) the targeted sequences.
  • the system contains a first atgRNA and a second atgRNA (see FIG.5)
  • the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, where the at least first pair of atgRNAs have domains that are capable of guiding the gene editor polypeptide or prime editor fusion protein to a target sequence
  • the first atgRNA further includes a first RT template that comprises at least a portion of the first integration recognition site
  • the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the first atgRNA’s reverse transcriptase template encodes for a first single-stranded DNA sequence (i.e., a first DNA flap) that contains a complementary region to a second single-stranded DNA sequence (i.e., a second DNA flap) encoded by a second atgRNA comprising a second reverse transcriptase template.
  • the complementary region between the first and second single-stranded DNA sequences is comprised of more than 5 consecutive bases of an integrase target recognition site.
  • the complementary region between the first and second single-stranded DNA sequences is comprised of more than 10 consecutive bases of an integrase target recognition site.
  • the complementary region between the first and second single-stranded DNA sequences is comprised of more than 20 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 30 consecutive bases of an integrase target recognition site.
  • Use of two guide RNAs that are (or encode DNA that is) partially complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site-containing guide RNAs (atgRNAs).
  • RNAs that are (or encode DNA that is) full complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site- containing guide RNAs (atgRNAs).
  • AtgRNAs As shown in FIGs. 1A-1E, AttP variants (SEQ ID NOS: 394 and 540-542, respectively, in order of appearance) can be optimized.
  • Table 9 includes atgRNAs, sgRNAs and nicking guides that can be used herein.
  • a gene editor polypeptide described herein contains an integrase or recombinase.
  • the integrase is delivered as a protein or the integrase is encoded in a delivered polynucleotide.
  • the integration enzyme is selected from the group consisting of Dre, Vika, Bxb1, ⁇ C31, RDF, ⁇ BTl, R1, R2, R3, R4, R5, TP901-1, A118, ⁇ FCl, ⁇ C1, MR11, TG1, ⁇ 370.l, W ⁇ , BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, ⁇ RV, retrotransposases encoded by a Tc1/mariner family member including but not limited to retro
  • Xu et al describes methods for evaluating integrase activity in E. coli and mammalian cells and confirmed at least R4, ⁇ C31, ⁇ BT1, Bxb1, SPBc, TP901-1 and W ⁇ integrases to be active on substrates integrated into the genome of HT1080 cells (Xu et al., 2013, Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol.2013 Oct 20;13:87. doi: 10.1186/1472-6750-13-87).
  • Durrant describes new large serine recombinases (LSRs) divided into three classes distinguished from one another by efficiency and specificity, including landing pad LSRs which outperform wild-type Bxb1 in episomal and chromosomal integration efficiency, LSRs that achieve both efficient and site- specific integration without a landing pad, and multi-targeting LSRs with minimal site-specificity. Additionally, embodiments can include any serine recombinase such as BceINT, SSCINT, SACINT, and INT10 (see Ionnidi et al., 2021; Drag-and-drop genome insertion without DNA cleavage with CRISPR directed integrases.
  • LSRs serine recombinases
  • the integration site can be selected from an attB site, an attP site, an attL site, an attR site, a lox71 site a Vox site, or a FRT site.
  • desired activity of integrases, transposases and the like can depend on nuclear localization.
  • prokaryotic enzymes are adapted to modulate nuclear localization.
  • eukaryotic or vertebrate enzymes are adapted to modulate nuclear localization.
  • the disclosure provides fusion or hybrid proteins.
  • Such modulation can comprise addition or removal of one or more nuclear localization signal (NLS) and/or addition or removal of one or more nuclear export signal (NES).
  • NLS nuclear localization signal
  • NES nuclear export signal
  • the one or more integration recognition sites is specific for a serine integrase other than BxB1.
  • the integration enzyme is selected from one of the about 27,000 Serine integrases described in International Patent Publication No. WO 2023/070031A2, which is hereby incorporated by reference in its entirety. 6.9. Split Gene Editor Guide RNA Compositions [0303] The present disclosure contemplates a split guide RNA comprised of two or more polynucleotides that are capable of forming a guide RNA complex (see Liu et al; doi: 10.1038/s41587-022-01255-9 the entirety of which is incorporated by reference herein).
  • the guide RNA may be a guide RNA complex, wherein the guide RNA complex is comprised of at least two polynucleotide components.
  • the guide RNA complex is comprised of a first polynucleotide component and a second polynucleotide component.
  • the first polynucleotide component comprises a spacer complementary to a target first genomic site and a scaffold capable of binding to a DNA binding nickase
  • the second polynucleotide component comprises a reverse transcriptase template comprised of at least an integrase target recognition site, a primer binding site and a RNA- protein recruitment domain.
  • the RNA-protein recruitment domain is a MS2 hairpin.
  • at least one of the first polynucleotide component and the second polynucleotide component are circularized.
  • circularization is mediated by a ribozyme or ligase.
  • circularization is mediated by covalently or non-covalently linking the 5’ and 3’ termini.
  • the guide RNA or the guide RNA complex further comprises one or more of an RNA-protein recruitment domain, RNA-RNA recruitment domain, a transcriptional termination signal, a reverse transcription termination signal, an RNA ribozyme, or a chemical linker.
  • one or more guide RNA complex is comprised in one or more RNA polynucleotides or DNA polynucleotides. 6.10. Guide RNA Compositions for Dual Guide RNA Systems [0307]
  • the guide RNA or the guide RNA complex reverse transcriptase template encodes for a first single-stranded DNA sequence (i.e., a first DNA flap) that contains a complementary region to a second single-stranded DNA sequence (i.e., a second DNA flap) encoded by a second guide RNA comprised of a reverse transcriptase template.
  • the complementary region between the first and second single-stranded DNA sequences is comprised of more than 5 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 10 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single- stranded DNA sequences is comprised of more than 20 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 30 consecutive bases of an integrase target recognition site.
  • integrase target recognition site is an Bxb1 attB or attP sequence.
  • the att-site central dinucleotide is not GT or CA.
  • one or more guide RNA or the guide RNA complex is comprised in one or more RNA polynucleotides or DNA polynucleotides.
  • the one or more guide RNA complex is a split guide RNA complex or split guide RNA system.
  • the one or more guide RNA complex is a split atgRNA complex or split atgRNA system.
  • a non-limiting example of a dual gRNA i.e., dual atgRNA system is as described in FIG.5. 6.11.
  • pleiopluripotent cells are edited using, at least in part, the information provided in FIGs.1-5.
  • editing the pleiopluripotent cell genome results in a cell that, following differentiation and transplantation into a subject, is capable of evading a subject’s immune system (e.g., encoding for one or more exogenous polypeptides that modulate immune response) but safeguarded from runaway expansion (e.g., inducible suicide genes) (see, e.g., FIG. 2).
  • immune system e.g., encoding for one or more exogenous polypeptides that modulate immune response
  • runaway expansion e.g., inducible suicide genes
  • the methods described herein are designed so that beacons (and ultimately the donor polynucleotide templates) are targeted to genomic loci with the aim of reducing expression (e.g., knocking out) of the gene associated with the particular genomic locus being targeted while also serving as a genomic locus suitable for expression of the aforementioned transgenes (e.g., exogenous polypeptides capable of modulating an immune response, inducible suicide genes, or a combination thereof).
  • the pleiopluripotent cell genome can be further modified to include additional integration recognition sites (i.e., landing pads and beacons) incorporated into the genome for integrating additional polynucleotides encoding therapeutic agents.
  • Example 10 provides a non-limiting exemplary method for generating pleiopluripotent stem cells having a beacon (integration recognition site) in each of the B2M, CIITA, TRAC, and AAVS1 loci.
  • the pleiopluripotent stem cells can then be used for programmable gene insertion. In some cases, this achieved using multiplex programmable gene insertion, for example, where a different donor polynucleotide template is inserted into each beacon site-specifically placed into the aforementioned loci (see, e.g., Example 11).
  • pleiopluripotent cells can be differentiated to a non-pleiopluripotent cell (e.g., a hematopoietic cell, a neuronal cell, a cardiac cell, or a pancreatic cell) and used as a source of material for allogeneic cell therapy.
  • a method of generating pleiopluripotent cells includes site- specifically incorporating at least a first integration recognition site into the genome of a pleiopluripotent cell.
  • site-specifically incorporating the at least first integration recognition site is effected by introducing into the pleiopluripotent stem cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein or a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the pleiopluripotent stem cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site- specifically incorporated into the genome of the pleiopluripotent stem cell: (i
  • the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is a first atgRNA that further includes a first RT template that comprises at least a portion of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a second atgRNA that further includes a second RT template that comprises at least a portion of the first integration recognition site, wherein the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
  • the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is an atgRNA that further includes an RT template that comprises at least a portion of the first integration recognition site, wherein the atgRNA encodes the entirety of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a nicking gRNA.
  • the methods include incorporating a plurality of integration recognition sites into the genome of a pleiopluripotent cell.
  • the method includes incorporating two integration recognition sites, three integration recognition sites, four integration recognition sites, five integration recognition sites, six integration recognition sites, seven integration recognition sites, eight integration recognition sites, nine integration recognition sites, or ten or more integration recognition sites into the genome of a pleiopluripotent cell.
  • each of the additional integration recognition sites e.g., second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth or more
  • a method of generating pleiopluripotent cells includes (a) site- specifically incorporating at least a first integration recognition site into the genome of a pleiopluripotent cell, wherein site-specifically incorporating the at least first integration recognition site is effected by introducing into the pleiopluripotent stem cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein or a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the pleiopluripotent stem cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a
  • incorporation of the integration recognition site into the pleiopluripotent cell genome is performed concurrently with integration of the donor polynucleotide template into the pleiopluripotent cell genome.
  • an at least first integration recognition site is incorporated into the genome of the pleiopluripotent cell using a homology directed repair, homology-independent target insertion (HITI), retrotransposon-mediated integration, viral vector-mediated integration (e.g., lentivirus), transposon-mediated integration (e.g., sleeping beauty), and/or recombinase- mediated integration (e.g., serine recombinase fC31 integrase).
  • HITI homology-independent target insertion
  • retrotransposon-mediated integration e.g., viral vector-mediated integration (e.g., lentivirus), transposon-mediated integration (e.g., sleeping beauty), and/or recombinase- mediated integration (e.g., serine
  • a method of generating a pleiopluripotent cell includes integrating, into the genome of the pleiopluripotent cell at an at least first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a second generation pleiopluripotent cell
  • a method of generating a pleiopluripotent cell includes integrating, into the genome of the pleiopluripotent cell at an at least first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the at least one incorporated genomic integration recognition sites by the integrase; and (b) site- specifically incorporating a second integration recognition site into the genome of the pleiopluripotent cell, thereby producing a third generation pleiopluripotent cell.
  • a method of generating a pleiopluripotent cell includes integrating, into the genome of the pleiopluripotent cell at an at least the first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the at least one incorporated genomic integration recognition sites by the integrase; (b) site- specifically incorporating a second integration recognition site into the genome of the pleiopluripotent cell; and (c) integrating a second donor polynucleotide template into the pleiopluripotent cell genome at the
  • incorporation of the second integration recognition site into the pleiopluripotent cell genome is performed concurrently with integration of the second donor polynucleotide template into the pleiopluripotent cell genome.
  • an integration recognition site i.e., beacon
  • an integration recognition site is incorporated into a pleiopluripotent cell genome by delivering into the cell: (i) a gene editor protein (or a polynucleotide encoding a gene editor protein) or a prime editor fusion protein (or a polynucleotide encoding a prime editor fusion protein), wherein the gene editor protein or prime editor fusion protein is capable of incorporating the integration recognition site into the genome, and (ii) two guide RNAs (e.g., two atgRNAs), wherein each of the two guide RNA encodes all or part of an integrase target recognition site.
  • a gene editor protein or a polynucleotide encoding a gene editor protein
  • the method includes integrating a donor polynucleotide template into the pleiopluripotent cell genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target site, and an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the incorporated integration recognition site by the integrase, thereby producing a genetically modified pleiopluripotent cell (e.g., a second generation pleiopluripotent cell).
  • a genetically modified pleiopluripotent cell e.g., a second generation pleiopluripotent cell.
  • incorporation of the integration recognition site (i.e., beacon) into the pleiopluripotent cell genome is performed concurrently with integration of the donor polynucleotide template into the pleiopluripotent cell genome.
  • an integration recognition site (i.e., beacon) is incorporated into a pleiopluripotent cell genome by delivering into the cell: (i) a gene editor protein (or a polynucleotide encoding a gene editor protein) or a prime editor fusion protein (or a polynucleotide encoding a prime editor fusion protein), wherein the gene editor protein or prime editor fusion protein is capable of incorporating the integration recognition site into the genome, and (ii) two guide RNA complexes (e.g., two complexes each comprising an atgRNA), wherein each of the two guide RNA complexes is comprised of at least one polynucleotide that encodes all or part of an integr
  • the method includes integrating a donor polynucleotide template into the pleiopluripotent cell genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target site, and an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the incorporated integration recognition site by the integrase, thereby producing a genetically modified pleiopluripotent cell (e.g., a second generation pleiopluripotent cell).
  • a genetically modified pleiopluripotent cell e.g., a second generation pleiopluripotent cell.
  • incorporation of the integration recognition site i.e., beacon/beacon placement
  • incorporation of the integration recognition site is performed concurrently with integration of the donor polynucleotide template into the pleiopluripotent cell genome.
  • an integration recognition site i.e., beacon/beacon placement
  • a gene editor protein or a polynucleotide encoding a gene editor protein
  • a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein
  • the gene editor protein or prime editor fusion protein is capable of incorporating the integration recognition site into the genome
  • one or more guide RNA wherein the one or more guide RNA encodes an integrase target recognition site (i.e., atgRNA), and (iii) optionally, a nicking guide RNA (ngRNA).
  • integrating a donor polynucleotide template into the pleiopluripotent cell genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target site, and an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the incorporated integration recognition site by the integrase, thereby producing a genetically modified pleiopluripotent cell (e.g., a second generation pleiopluripotent cell).
  • a genetically modified pleiopluripotent cell e.g., a second generation pleiopluripotent cell.
  • incorporation of the integration recognition site i.e., beacon/beacon placement
  • incorporation of the integration recognition site is performed concurrently with integration of the donor polynucleotide template into the pleiopluripotent cell genome.
  • an integration recognition site i.e., beacon/beacon placement
  • a gene editor protein or a polynucleotide encoding a gene editor protein
  • a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein
  • the gene editor protein or prime editor fusion protein is capable of incorporating the integration recognition site into the genome
  • one or more guide RNA complex wherein the one or more guide RNA complex is comprised of at least one polynucleotide that encodes an integrase target recognition site (i.e., atgRNA), and (iii) optionally, a nicking guide RNA (ngRNA).
  • integrating a donor polynucleotide template into the pleiopluripotent cell genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target site, and an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the incorporated integration recognition site by the integrase, thereby producing a genetically modified pleiopluripotent cell (e.g., a second generation pleiopluripotent cell).
  • a genetically modified pleiopluripotent cell e.g., a second generation pleiopluripotent cell.
  • incorporation of the integration recognition site (i.e., beacon/beacon placement) into the pleiopluripotent cell genome is performed concurrently with integration of the donor polynucleotide template into the pleiopluripotent cell genome.
  • the pleiopluripotent cell is a pluripotent stem cell.
  • the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
  • the pleiopluripotent cell is a human cell. 6.11.1.
  • the pleiopluripotent comprises a genetic perturbation that results in reduction of an allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell.
  • at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell.
  • the pleiopluripotent cell includes a plurality of integration recognition sites at least one of which is incorporated into the pleiopluripotent cell genome at a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell.
  • loci disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell, are as described in U.S. Patent No. 10,968,426, which is herein incorporated by reference in its entirety.
  • the pleiopluripotent stem cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
  • the deletion or reduced expression results from knockout or knockdown using base editing, prime editing, other CRISPR/Cas9-mediated methods, TALE- based methods, Zinc Finger Nuclease-based methods, siRNA, miRNA, RNAi, or a combination thereof.
  • the pleiopluripotent stem cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof as the result of knockout or knockdown of genes expressed from one or more chromosomal regions selected from: HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA- DQ.
  • the pleiopluripotent stem cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof, as a result of incorporation of one or more integration recognition sites into a genomic loci that when disrupted leads to deletion or reduced expression of the associated gene.
  • At least one of the at least first integration recognition sites is incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
  • the pleiopluripotent cell has reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
  • incorporation of the at least first integration recognition site results in reduction of a potential allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell (when administered to a patient in need thereof).
  • At least one of the at least first integration recognition sites is incorporated into the genome at a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
  • the pleiopluripotent cell has reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
  • incorporation of the at least first integration recognition site results in reduction of a potential allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell (when administered to a patient in need thereof).
  • At least one of the at least first integration recognition sites is incorporated into the genome at a locus selected from B2M locus, CIITA locus, HLA-A locus, HLA- B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus.
  • a locus selected from B2M locus, CIITA locus, HLA-A locus, HLA- B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3
  • incorporation of the at least first integration recognition site results in reduction of a potential allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell (when administered to a patient in need thereof).
  • at least one of the at least first integration recognition sites is incorporated into the genome at the B2M locus. In such cases, incorporation of the at least first integration recognition site at the B2M locus results in reduction of a potential allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell (when administered to a patient in need thereof).
  • At least one of the at least first integration recognition sites is incorporated into the genome at the CIITA locus.
  • incorporation of the at least first integration recognition site at the CIITA locus results in reduction of a potential allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell (when administered to a patient in need thereof).
  • At least one of the at least first integration recognition sites is incorporated into the genome at locus associated with blood group antigens, disruption of which is capable of reducing expression of the blood group antigens by the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell.
  • at least one of the at least first integration recognition sites is incorporated into the genome at an alleles of the ABO gene (alpha 1-3-N-acetylgalactosaminyltransferase and alpha 1-3-galactosyltransferase), including, without limitation ABO*A1.01 and ABO* B.01.
  • the pleiopluripotent cell is an iPSC derived from a healthy donor in order to minimize immunity against ABO antigens.
  • the pleiopluripotent cell is an iPSC derived from a healthy donor with blood type O. 6.11.2.
  • an integration recognition site is incorporated (i.e., beacon placement) into a pleiopluripotent cell genome using a single atgRNA and a single nicking guide RNA (ngRNA) (see, e.g., FIG. 5).
  • ngRNA nicking guide RNA
  • an integration recognition site is incorporated into a pleiopluripotent cell genome using two atgRNAs (dual atgRNAs) (see, e.g., FIG. 5).
  • Beacon placement efficiency can be determined, for example, by a two-color digital droplet PCR assay that compares signal from no-insertion amplicons (i.e., wild type) with signal from beacon inserted/placed amplicons. Beacon placement efficiency can be determined, for example, by amplicon-sequencing (amp-seq).
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 8
  • At least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a locus encoding an exon.
  • the exon is coding sequence of a gene of interest, wherein incorporation of the integration recognition site into the exon is capable of reducing or deleting expression of the gene of interest.
  • incorporation of integration recognition site at one or more exons associated with a gene of interest results in reduced expression of at least 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the gene of interest as compared to a cell where the integration recognition site is not incorporated at one or more exons associated with the gene of interest.
  • efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at an exon of a gene of interest depends on the molar ratio of a single atgRNA and a single ngRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 900 picomoles total of atgRNA plus ngRNA.
  • an integration recognition site is incorporated into a pluripotent cell genome at an exon of a gene of interest using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 100 to about 1000 picomoles total of atgRNA plus ngRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein).
  • efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at an exon of a gene of interest depends on the molar ratio of each atgRNA in a dual atgRNA method.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest using dual atgRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of each atgRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest using dual atgRNAs at a 1:1 molar ratio of each atgRNA at about 100 to about 1000picomoles total of atgRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest using dual atgRNAs at a 1:1 molar ratio of each atgRNA at about 100 to about 1000 picomoles total of atgRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein).
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
  • the integration recognition site is incorporated at one or more loci associated with HLA Class I proteins, one or more loci associate with HLA Class II proteins, or a combination thereof. In some embodiments, the integration recognition site is incorporated at one or more loci associated with HLA Class I proteins. In some embodiments, the integration target site is incorporated at one or more loci associated with HLA Class II proteins. [0345] In some embodiments, incorporation of integration recognition site at one or more loci associated with HLA Class I proteins results in reduced expression of the one or more HLA Class I proteins as compared to a cell where the integration recognition site is not incorporated at one or more loci associated with HLA Class I proteins.
  • incorporation of integration recognition site at one or more loci associated with HLA Class I proteins results in reduced expression of at least 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% for each of the one or more HLA Class I proteins as compared to a cell where the integration recognition site is not incorporated at one or more loci associated with HLA Class I proteins.
  • incorporation of integration target recognition at one or more loci associated with HLA Class II proteins results in reduced expression of the one or more HLA Class II proteins as compared to a cell where the integration recognition site is not incorporated at one or more loci associated with HLA Class II proteins.
  • incorporation of integration recognition site at one or more loci associated with HLA Class II proteins results in reduced expression of at least 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% for each of the one or more HLA Class II proteins as compared to a cell where the integration recognition site is not incorporated at one or more loci associated with HLA Class II proteins.
  • the integration recognition site is incorporated at one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
  • the integration recognition target site is incorporated at one or more of the B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus.
  • a donor polynucleotide is integrated at one or more incorporated integration target site.
  • efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at the B2M locus depends on the molar ratio of a single atgRNA and a single ngRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 900 picomoles total of atgRNA plus ngRNA.
  • an integration recognition site is incorporated into a pluripotent cell genome at the B2M locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 100 to about 1000 picomoles total of atgRNA plus ngRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein).
  • efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at the B2M locus depends on the molar ratio of each atgRNA in a dual atgRNA method.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus using dual atgRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of each atgRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus using dual atgRNAs at a 1:1 molar ratio of each atgRNA at about 100 to about 1000picomoles total of atgRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus using dual atgRNAs at a 1:1 molar ratio of each atgRNA at about 100 to about 1000 picomoles total of atgRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein).
  • efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at the CIITA locus depends on the molar ratio of a single atgRNA and a single ngRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 900 picomoles total of atgRNA plus ngRNA.
  • an integration recognition site is incorporated into a pluripotent cell genome at the CIITA locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 100 to about 1000 picomoles total of atgRNA plus ngRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein).
  • efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at the CIITA locus depends on the molar ratio of each atgRNA in a dual atgRNA method.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus using dual atgRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of each atgRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus using dual atgRNAs at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of each atgRNA at about 100 to about 1000 picomoles total of atgRNA.
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus using dual atgRNAs at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of each atgRNA at about 100 to about 1000 picomoles total of atgRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein).
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
  • an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
  • an integration recognition site is specific for an integrase.
  • the integration recognition site is specific for a recombinase or transposase.
  • the integration recognition site is an attB or attP site.
  • the integration recognition site is a modified or orthogonal attB or attP site.
  • the integrase target recognition site is a Bxb1 attB or attP sequence.
  • the att-site central dinucleotide is not GT or CA.
  • the integration recognition site is comprised of 38 or 46 nucleotides.
  • the integration recognition site is less than 46 nucleotides. In some embodiments, the integration recognition site is less than 40 nucleotides. In some embodiments, the integration recognition site is less than 35 nucleotides. In some embodiments, the integration recognition site is less than 30 nucleotides. In some embodiments, the integration recognition site is less than 25 nucleotides. [0358] In some embodiments, the one or more guide RNA or the one or more guide RNA complex comprises a chemical modification. In certain embodiments, the chemical modification is selected from one or more of a 2’ O-methyl and phosphorothioate.
  • RNAs i.e., attachment site-containing guide RNAs
  • Table 12 includes dual atgRNAs for incorporating an integration recognition site at the B2M locus or CIITA locus.
  • the guide RNA (e.g., atgRNA) for beacon placement comprises, in 5’ to 3’ order, (i) a spacer complementary to a first target genomic site, (ii) a scaffold capable of binding to a DNA binding nuclease or nuclease with nickase activity (or cleaves activity), (iii) a reverse transcriptase template comprised of at least an integrase target recognition site (e.g., all or a portion of an integrase target recognition site), and (iv) a primer binding site.
  • the reverse transcriptase template encodes for a first single-stranded DNA sequence or first DNA flap sequence.
  • a cellular integrated polynucleotide encodes for one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, and one or more adapters (tags), or a combination thereof. (See, e.g., FIG.3A).
  • a cellular integrated polynucleotide i.e., donor polynucleotide template
  • the one or more integration recognition sites encoded in the cellular integrated polynucleotide can be used to integrate a second cellular integrated polynucleotide (e.g., a second donor polynucleotide template) in the genomic loci where the first cellular integrated polynucleotide integrated.
  • the one or more integration recognition sites encoded in the cellular integrated polynucleotide i.e., donor polynucleotide template
  • expression of the one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof is driven by a pleiopluripotent cell-specific regulatory elements (e.g., a promoter, an enhancer, or a combination thereof).
  • pleiopluripotent cell-specific regulatory elements e.g., a promoter, an enhancer, or a combination thereof.
  • Non-limiting examples of pleiopluripotent cell-specific promoters include a Nanog promoter, a Oct4 promoter, an hTERT promoter, and a Sox2 promoter.
  • expression of the one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof is driven by a constitutive promoter.
  • the first donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 1 kilobase (kb), at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, at least 30kb, at least 1 kilobase (kb), at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at
  • the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 5 kilobases (kb). In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 10 kilobases (kb). In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 15 kilobases (kb). In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 20 kilobases (kb).
  • the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 25 kilobases (kb). In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 30 kilobases (kb). In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 35 kilobases (kb).
  • the first donor polynucleotide template integrated into the pleiopluripotent cell genome is at least at least 1 kilobase (kb), at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, at least 30kb, at least 31kb, at least 32kb, at least 33kb, at least 34kb, or at least 35kb or more.
  • the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 5 kilobases (kb). In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 10 kilobases (kb). In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 15 kilobases (kb). In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 20 kilobases (kb).
  • the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 25 kilobases (kb). In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 30 kilobases (kb). In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 35 kilobases (kb). 6.11.3.1 Inducible Suicide Genes [0369] In certain embodiments, the cellular integrated polynucleotide (i.e., donor polynucleotide template) encodes for one or more inducible suicide genes.
  • the term “inducible suicide gene” refers to an engineered polypeptide designed to prevent the adverse events of cell therapy.
  • the inducible suicide gene is conditionally controlled to address adverse events of cell therapy (e.g., safety concerns).
  • Conditional regulation can include control through a small molecule-mediated post-translational activation, tissue-specific regulation, temporal transcriptional regulation, or a combination thereof.
  • the inducible suicide gene could mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation and/or antibody-mediated depletion.
  • the inducible suicide gene is activated by an exogenous molecule, e.g., a prodrug, that when activated, triggers apoptosis and/or cell death of a therapeutic cell.
  • an exogenous molecule e.g., a prodrug
  • inducible suicide genes include to suicide genes such as caspase 9 (or caspase 3 or 7), herpes simplex virus thymidine kinase (HSV-TK), cytosine deaminase, modified EGFR, nitroreductase, purine nucleoside phosphorylase, horseradish peroxidase, a HER1 transgene, a RQR8 transgene, or a combination thereof.
  • a prodrug e.g., an exogenous molecule
  • the one or more inducible suicide genes is caspase9.
  • the one or more inducible suicide genes comprises two caspase9 proteins where each caspase9 protein is operably linked to a different promoter sequence (See FIG. 3A). Relying on two different promoters enables both time and tissue specific control over expression of each caspase9 protein.
  • each caspase 9 protein is fused to a different controllable domain (e.g., FKBP or mFRM and FKBP), thereby generating controllable caspase9 proteins.
  • a controllable caspase9 protein is controlled using an exogenous molecule.
  • the exogenous molecule is AP20187 (or an analog thereof).
  • AP20187 enables homodimerization of Caspase9-FKBP fusions.
  • FIG.4 illustrates caspase9-FKBP fusions where expression is driven by a nanog promoter.
  • the caspase9 FKBP protein is expressed while the cells are in a pluripotent state. Contacting the cell with AP20187 during the pluripotent state results in cell death.
  • the exogenous molecule is AP21967 (or an analog thereof).
  • FIG.4 illustrate caspase9-FKBP and caspase9-mFRB where expression is driven by a beta-actin promoter (or another constitutively active promoter).
  • the Caspase9-FKBP and Caspase9-mFRB fusion are expressed constitutively. Therefore, contacting the cells with the AP21967 results in cell death.
  • Either AP20187 and/or AP21967 can be administered to a subject who was previously been administered cells containing the HSV-TK gene.
  • the one or more inducible suicide genes is herpes simplex virus thymidine kinase (HSV-TK).
  • HSV-TK converts ganciclovir into a toxic product and allows selective elimination of cells expressing the HSV-TK gene.
  • Ganciclovir can be administered to a subject who was previously been administered cells containing the HSV-TK gene. In such cases, ganciclovir is used to eliminate the previously administered cells.
  • the donor polynucleotide templates comprise three suicide genes, including a first caspase9 protein fused to a FKBP domain operably linked to a Nanog promoter, a second caspase9 protein used to a mFRB domain and a FKBP domain operably linked to a Beta- actin promoter, and a thymidine kinase (see, e.g. FIG. 3A).
  • each of the inducible suicide genes is operably linked to a promoter.
  • the thymidine kinase is linked to one of the caspase9 proteins by a 2A self-cleaving peptide (e.g., a P2A, E2A,T2A, and F2A) or an internal ribosome entry site (IRES) (see, e.g., FIG.3A).
  • a 2A self-cleaving peptide e.g., a P2A, E2A,T2A, and F2A
  • IRS internal ribosome entry site
  • integrating a donor polynucleotide template encoding one or more inducible suicide genes into the pleiopluripotent cell genome at the one or more incorporated target recognition sites results in increased expression of the one or more inducible suicide genes as compared to a pluripotent stem genome not having a donor polynucleotide template integrated into the cell’s genome at the one or more incorporated target recognition sites.
  • integrating a donor polynucleotide template encoding one or more inducible suicide genes into the pleiopluripotent cell genome at the one or more incorporated target recognition sites results in increased expression of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more of the one or more inducible suicide genes as compared to a pluripotent stem genome not having a donor polynucleotide template integrated into the cell’s genome at the one or more incorporated target recognition sites.
  • the encoded one or more inducible suicide genes further comprises an integrase target recognition site.
  • the encoded one or more inducible suicide genes further comprises at least two integrase target recognition sites.
  • the one or more integrase target recognition sites flank the encoded one or more inducible suicide genes.
  • the flanking integrase target recognition sites allow for excision of the one or more inducible suicide genes. 6.11.3.2 Exogenous Polypeptides Capable of Modulating an Immune Response [0375]
  • the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells or cells derived therefrom.
  • the one or more exogenous polypeptides capable of modulating an immune response prevent an immune response to the pleiopluripotent cells (or cells derived therefrom) by inhibiting, blocking, or preventing recognition of the pleiopluripotent cell by macrophages, T-cells, Natural Killer (NK)-cells, antigen-presenting cells, or a combination thereof.
  • the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL-1, HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof.
  • integrating a donor polynucleotide template encoding one or more exogenous polypeptides capable of modulating an immune response into the pleiopluripotent cell genome at the one or more incorporated target recognition sites results in increased expression of the one or more polypeptides capable of modulating an immune response as compared to a pluripotent stem genome not having a donor polynucleotide template integrated into the cell’s genome at the one or more incorporated target recognition sites.
  • integrating a donor polynucleotide template encoding one or more exogenous polypeptides capable of modulating an immune response into the pleiopluripotent cell genome at the one or more incorporated target recognition sites results in increased expression of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more of the one or more inducible suicide genes as compared to a pluripotent stem genome not having a donor polynucleotide template integrated into the cell’s genome at the one or more incorporated target recognition sites.
  • the cellular integrated polynucleotide encodes for two or more exogenous polypeptides capable of modulating an immune response where the sequence encoding the two or more exogenous polypeptides are separated by a 2A self-cleaving peptide (e.g., a P2A, E2A,T2A, and F2A).
  • a 2A self-cleaving peptide e.g., a P2A, E2A,T2A, and F2A
  • the sequences encoding the two or more exogenous polypeptides are separated by an internal ribosome entry site (IRES).
  • a cellular integrated polynucleotide includes: CD47, PDL-1, and B2M-HLA-E.
  • the sequences encoding CD47, PDL-1 and B2M- HLA-E are separated by either a 2A self-cleaving peptide (e.g., a P2A, E2A,T2A, and F2A) or an IRES sequence.
  • a 2A self-cleaving peptide e.g., a P2A, E2A,T2A, and F2A
  • IRES sequence A non-limiting example of a cellular integrated polynucleotide is described in FIG.3A.
  • the encoded one or more exogenous polypeptides capable of modulating an immune response further comprises an integrase target recognition site.
  • the encoded one or more exogenous polypeptides capable of modulating an immune response further comprises at least two integrase target recognition sites.
  • the one or more integrase target recognition sites flank the encoded one or more exogenous polypeptides capable of modulating an immune response. In certain embodiments, the flanking integrase target recognition sites allow for excision of the one or more exogenous polypeptides capable of modulating an immune response. [0380] In some embodiments, the one or more exogenous polypeptides capable of modulating an immune response are capable of serving as a safety switch target. In some embodiments, the one or more exogenous polypeptides can be targeted with an antibody that is capable of specifically binding to the one or more exogenous polypeptides, thereby blocking the polypeptides ability to prevent an immune response to the pleiopluripotent cells, or cells derived therefrom.
  • a pleiopluripotent cell comprising an exogenous CD47 on it surface can be contacted with an anti- CD47 antibody, wherein the anti-CD47 antibody blocks CD47 interaction with macrophage signal regulatory protein (SIRP ⁇ ), thereby allowing recognition and elimination of the cell by macrophages.
  • SIRP ⁇ macrophage signal regulatory protein
  • the pleiopluripotent cell comprising an exogenous PD-L1 on it surface can be contacted with an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody blocks PD-L1 interaction with PD-1, thereby allowing recognition and elimination of the cell by T cells.
  • a first or second cellular integrated polynucleotide (i.e., a first or second donor polynucleotide template) includes a sequence encoding a tag that enables isolation of the pleiopluripotent cell.
  • the tag can be a stand-alone molecule such as a polypeptide expressed on the surface of the pleiopluripotent cell or a cell-derived from the pleiopluripotent cell.
  • stand-alone tags includes non-natively expressed receptors (e.g., nerve truncated growth factor receptor and truncated nerve growth factor receptor).
  • the tag can be an amino acid sequence fused to an amino acid sequence of an endogenous protein that is expressed by a pleiopluripotent cell or a cell-derived from the pleiopluripotent cell.
  • tags that can be used to isolate a cell having a first or second cellular integrated polynucleotide include an ALFA tag system (e.g., SRLEEELRRRLTE (SEQ ID NO: 615)).
  • the ALFA tag is fused to the N-terminus of CD47 (see FIG.3A).
  • the first or second cellular integrated polynucleotide includes a sequence encoding an ALFA tag fused to one or more polypeptide expressed on the surface of the cell (e.g., one of more of the exogenous polypeptides capable of modulating an immune response).
  • a nanobody designed to bind specifically to the ALFA can be used to bind and isolate the pleiopluripotent cells having the tag on the surface.
  • an ALFA tag can be used to identify pleiopluripotent cells that include the first and/or second cellular integrated polynucleotide integrated into its genome.
  • an ALFA nanobody (NbALFA) is fused to one or more polypeptides on the surface of the cell, one or more ALFA tagged polypeptides can be used to engage with NbALFA fusion polypeptides for therapeutic purposes. 6.11.4.
  • an additional integration recognition site is incorporated into a pleiopluripotent cell genome by delivering into the cell: (i) a gene editor protein (or a polynucleotide encoding a gene editor protein) or a prime editor fusion protein (or a polynucleotide encoding a prime editor fusion protein), wherein the gene editor protein or prime editor fusion protein is capable of incorporating the integration recognition site into the genome, (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the pleiopluripotent stem cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site
  • RT reverse transcriptase
  • the additional integration recognition site is incorporated into the pleiopluripotent cell genome at a locus encoding an exon.
  • the exon is coding sequence of a gene of interest, wherein incorporation of the integration recognition site into the exon is capable of reducing or deleting expression of the gene of interest.
  • the additional integration recognition is incorporated into the pleiopluripotent cell genome at a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent cell.
  • Non-limiting examples of loci, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent cell, are described herein.
  • Incorporating an additional integration recognition site into a pleiopluripotent cell genome enables subsequent round of genetic modification of the pleiopluripotent cell genome.
  • a second donor polynucleotide can be integrated at the genomic locus where the additional integration recognition sites were incorporated in the early round of editing.
  • a genetically modified pleiopluripotent cell comprises one, two, three, four, five, six, seven, eight, nine, ten, or more integration recognition sites.
  • the at least one, at least two, at least three, at least four, at least five or more of the integration recognition sites can be used for placing a second donor polynucleotide template into the pleiopluripotent cell genome. 6.11.5.
  • a second donor polynucleotide template e.g., a second cellular integrated polynucleotide
  • a second cellular integrated polynucleotide i.e., second donor polynucleotide template
  • an additional integration recognition site e.g., any of the additional integration recognition sites described herein.
  • a second donor polynucleotide template encodes transcription factors, receptors, chimeric antigen receptors, signaling molecules, secreted proteins, cytokines, enzymes, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, and persistence and/or survival of the iPSCs or cells derived from the iPSC.
  • a second donor polynucleotide template encodes for one or more of a chimeric antigen receptor, a T cell receptor, a cytokine receptor, a chemokine receptor, or a modified receptor.
  • the second donor polynucleotide template encodes for an exogenous T cell receptor. In some embodiments, the second cellular integrated polynucleotide encodes for a secreted cytokine. In some embodiments, the second donor polynucleotide template encodes for a recombinant protein.
  • the second donor polynucleotide template further comprises one or more of a co-stimulatory receptor such suitable co-stimulatory signaling regions are also well known in the art, and include members of the B7/CD28 family such as B7-1, B7-2, E7-H1, B7-H2, E7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2 or PDCD6; or ILT/CD85 family proteins such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3 or LILRB4; or tumor necrosis factor (TNF) superfamily members such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, OX40, RELT, TACI, TL1A, TNF-alpha or TNF RII;
  • the selection of the co-stimulatory signaling regions may be selected depending upon the particular use intended for the transformed cells.
  • the co-stimulatory signaling regions selected for those which may work co-operatively or synergistically together may be selected from CD28, CD27, ICOS, 4-1BB, OX40, CD30, GITR, HVEM, DR3 or CD40.
  • the encoded one or more chimeric antigen receptor, T cell receptor, cytokine receptor, chemokine receptor, or modified receptor further comprises one or more integrase target recognition site.
  • the encoded one or more chimeric antigen receptor, T cell receptor, cytokine receptor, chemokine receptor, or modified receptor further comprises at least two integrase target recognition sites.
  • the one or more integrase target recognition sites flank the encoded one or more chimeric antigen receptor, T cell receptor, cytokine receptor, chemokine receptor, or modified receptor.
  • the flanking integrase target recognition sites allow for excision of any inserted genetic sequence.
  • a first or second donor polynucleotide template encodes one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
  • the first or second donor polynucleotide template encodes one or more HLA Class I proteins or one or more HLA Class II proteins that has reduced expression (e.g., due to a mutation or due to genetic ablation using a knockdown-mediated technique) in the pleiopluripotent cell or cell differentiated from the pleiopluripotent cell.
  • the HLA Class 1 protein is a B2M protein.
  • a first or second donor polynucleotide template includes an inducible promoter that controls expression of the one or more HLA Class I proteins, the one or more HLA Class II proteins, or a combination thereof.
  • the one or more HLA Class I proteins or one or more HLA Class II proteins can be “switched-on” at a later time point.
  • inducible promoter sequences that can be used in the methods described herein include a tetracycline-inducible promoter, an alcohol regulated promoter (e.g., AlcA promoter and AlcR activator), and steroid-regulated promoter (e.g., a LexA promoter and a XVE activator), a temperature-inducible promoter (e.g., Hsp70 or Hsp90), and a light-inducible promoter (e.g., FixK2 promoter and blue-light sensing protein YFI).
  • an alcohol regulated promoter e.g., AlcA promoter and AlcR activator
  • steroid-regulated promoter e.g., a LexA promoter and a XVE activator
  • Hsp70 or Hsp90 e.g., Hsp70
  • the inducible promoter is a tetracycline inducible promoter.
  • a non-limiting example of a donor polynucleotide template that can be incorporated into the genome of a pleiopluripotent cell using the methods described herein includes the donor polynucleotide templates are as described in FIGs.3A-3B, FIG.17, and FIG.18 (see also SEQ ID NOs: 616 and 617).
  • a first or second cellular integrated polynucleotide i.e., a first or second donor polynucleotide template
  • a first or second donor polynucleotide template that includes an inducible promoter that controls expression of the one or more HLA Class I proteins, the one or more HLA Class II proteins, or a combination thereof, is incorporated into a pleiopluripotent cell’s genome at locus that allows for inducible gene expression can be “switched-on” at a later time point.
  • Non-limiting criteria for genomic loci that enable inducible gene expression at a later time point include those that remain accessible to transcriptional machinery (i.e., capable of being expressed) when the cell is in a pluripotent state as well as when the cells are differentiated to one or more of the cell types described herein (e.g., a hematopoietic cell, a neuronal cell, a cardiac cell, and a beta-cell).
  • a non- limiting example of a genomic loci that fits this criteria is the CIITA locus. 6.11.6.
  • an integration recognition site i.e., beacon
  • the donor polynucleotide template is integrated into the pleiopluripotent cell genome at the safe harbor loci or the genomic safe harbor.
  • an additional integration recognition site e.g., a second integration recognition site is incorporated into a pleiopluripotent cell genome at a safe harbor locus or a genomic safe harbor.
  • the second donor polynucleotide template described herein is integrated into the pleiopluripotent cell genome at the safe harbor loci or the genomic safe harbor.
  • Safe harbor loci, safe harbor site, and genomic safe can be used interchangeably.
  • a safe harbor locus is region of the human genome (e.g., intragenic or extragenic regions) that are able to accommodate predictable expression of newly integrated DNA without adverse effects on the host cell or organism.
  • a useful safe harbor must permit sufficient transgene expression to yield desired levels of the proteins encoded in the donor template polynucleotide.
  • a safe harbor also must not predispose cells to malignant transformation nor alter cellular functions.
  • Non-limiting examples of criteria that describe a safe harbor loci and/or genomic safe harbor include (i) absence of disruption of regulatory elements or genes, as judged by sequence annotation; is an intergenic region in a gene dense area, or a location at the convergence between two genes transcribed in opposite directions; (ii) keep distance to minimize the possibility of long-range interactions between vector-encoded transcriptional activators and the promoters of adjacent genes, particularly cancer-related and microRNA genes; and (iii)ubiquitous transcriptional activity, as reflected by broad spatial and temporal expression patterns (e.g., as measured by expression analysis), chromatin accessibility patterns (e.g., as measured by ATAC-seq), or methylation patterns (e.g., as measured by whole- genome bisulfite sequencing), indicating ubiquitous transcriptional activity.
  • broad spatial and temporal expression patterns e.g., as measured by expression analysis
  • chromatin accessibility patterns e.g., as measured by ATAC-seq
  • methylation patterns e.g., as
  • genomic safe harbor it is important for the safe harbor loci or genomic safe harbor to have the potential for ubiquitous expression because pleiopluripotent cells during differentiation experience changes in chromatin accessibility and/or remodeling that can lead to silencing of some loci and potential activation of others.
  • genomic safe harbor include those as described in Aznauryan et al. (Cell Reports Methods, Discovery and validation of human genomic safe harbor sites for gene and cell therapies, 2(1): 100154 (2022); see Table S1, including Genomic coordinates: chromosome 1: 195338589-195818588 and chromosome 3: 22720711-22761389).
  • AAVS1 adeno-associated virus site 1
  • CCR5 chemokine receptor 5
  • CCR5 chemokine receptor 5
  • a frequently used human SHS is the AAVS1 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion.
  • Another locus comprises the human homolog of the murine Rosa26 locus.
  • Yet another SHS comprises the human H11 locus on chromosome 22. In some cases, a complete gene may be prohibitively large and replacement of an entire gene impractical. 6.11.7.
  • compositions and methods for correcting or replacing genes or gene fragments (including introns or exons) or inserting genes in new locations comprises recombination or integration of a therapeutic agent into a safe harbor site (SHS) (e.g., any of the safe harbor sites described herein).
  • a method of the invention comprises recombining corrective gene fragments into a defective locus.
  • the methods and compositions can be used to target, without limitation, pleiopluripotent cells, for example embryonic stem cells or induced pleiopluripotent cells (iPSCs) cells at various stages of potency.
  • iPSCs induced pleiopluripotent cells
  • correcting or replacing genes or gene fragment includes integrating the corrective gene or fragment (the therapeutic agent) into one or more additional integration recognition sites previously incorporated into the pleiopluripotent cell genome.
  • the genes and targets described herein are encoded in the first donor template nucleotide, the second donor template nucleotide, or both.
  • the genes and targets described herein are integrated into the pleiopluripotent cell genome at the corresponding loci in which the integration target recognition sequences have been integrated (e.g., the one or more integration recognition sites or the one or more additional integration recognition sites).
  • methods and compositions of the invention are adapted to differentiate muscle cells to cardiomyocytes for Duchene Muscular Dystrophy (DMD).
  • the dystrophin gene is the largest gene in the human genome, spanning ⁇ 2.3 Mb of DNA. DMD is composed of 79 exons resulting in a 14-kb full-length mRNA. Common mutations include mutations that disrupt the reading frame of generate a premature stop codon.
  • An aspect of DMD that lends it to gene editing as a therapeutic approach is the modular structure of the dystrophin protein. Redundancy in the central rod domain permits the deletion of internal segments of the gene that may harbor loss-of-function mutations, thereby restoring the open reading frame (ORFs).
  • Inherited Retinal Diseases • Stargardt Disease (ABCA4) • Leber congenital amaurosis 10 (CEP290) • X linked Retinitis Pigmentosa (RPGR) • Autosomal Dominant Retinitis Pigmentosa (RHO) Liver Diseases: • Wilson’s disease (ATP7B) • Alpha-1 antitrypsin (SERPINA1) Intellectual Disabilities: • Rett Syndrome (MECP2) • SYNGAP1-ID (SYNGAP1) • CDKL5 deficiency disorder (CDKL5) Peripheral Neuropathies: • Charcot-Marie-Tooth 2A (MFN2) Lung Diseases: • Cystic Fibrosis (CFTR) • Alpha-1 Antitrypsin (SERPINA1) Autoimmune diseases: • IgA Nephropathy (Berger’s disease) • Anti-Neutrophil Cytoplasmic
  • the most common cystic fibrosis (CF) mutation F508del removes a single amino acid.
  • recombining human CFTR into an SHS of a cell that expresses CFTR F508del is a corrective treatment path.
  • the methods and systems described herein are used to CF by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing CF. Proposed validation is detection of persistent CFTR mRNA and protein expression in transduced cells.
  • Sickle cell disease (SCD) is caused by mutation of a specific amino acid — valine to glutamic acid at amino acid position 6.
  • SCD is corrected by recombination of the HBB gene into a safe harbor site (SHS) and by demonstrating correction in a proportion of target cells that is high enough to produce a substantial benefit.
  • the methods and systems described herein are used to sickle cell disease by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the disease.
  • validation is detection of persistent HBB mRNA and protein expression in transduced cells.
  • DMD Duchenne Muscular Dystrophy. The dystrophin gene is the largest gene in the human genome, spanning ⁇ 2.3 Mb of DNA.
  • DMD is composed of 79 exons resulting in a 14- kb full-length mRNA. Common mutations include mutations that disrupt the reading frame of generate a premature stop codon.
  • An aspect of DMD that lends it to gene editing as a therapeutic approach is the modular structure of the dystrophin protein. Redundancy in the central rod domain permits the deletion of internal segments of the gene that may harbor loss-of-function mutations, thereby restoring the open reading frame (ORFs).
  • ORFs open reading frame
  • recombination will be into safe harbor sites (SHS).
  • a frequently used human SHS is the AAVS1 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion.
  • the site is the human homolog of the e murine Rosa26 locus (pubmed.ncbi.nlm.nih.gov/18037879). In some embodiments, the site is the human H11 locus on chromosome 22.
  • Proposed target cells for recombination include stem cells for example induced pluripotent stem cells (iPSCs) and cells at various stages of differentiation. In some cases, a complete gene may be prohibitively large and replacement of an entire gene impractical. In such instances, rescuing mutants by recombining in corrected gene fragments with the methods and systems described herein is a corrective option.
  • iPSCs induced pluripotent stem cells
  • correcting mutations in exon 44 (or 51) by recombining in a corrective coding sequence downstream of exon 43 (or 50), using the methods and systems described herein is a corrective option.
  • Proposed validation is detection of persistent DMD mRNA and protein expression in transduced cells.
  • correcting factor VIII deficiency by recombining the FVIII gene into an SHS is a corrective path.
  • the methods and systems described herein are used to correct factor VIII deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the FIX deficiency. Proposed validation is detection of persistent FVIII mRNA and protein expression in transduced cells.
  • Factor 9 (Factor IX) Hemophilia B, also called factor IX (FIX) deficiency is a genetic disorder caused by missing or defective factor IX, a clotting protein.
  • the methods and systems described herein are used to correct factor IX deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the FIX deficiency. Proposed validation is detection of persistent FiX mRNA and protein expression in transduced cells.
  • Ornithine transcarbamylase deficiency OTCD
  • Ornithine transcarbamylase deficiency is a rare genetic condition that causes ammonia to build up in the blood. The condition – more commonly called OTC deficiency — is more common in boys than girls and tends to be more severe when symptoms emerge shortly after birth.
  • the methods and systems described herein are used to correct OTC deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the OTC deficiency or integrates a polynucleotide encoding a functional ornithine transcarbamylase enzyme.
  • Proposed validation is detection of persistent OTC mRNA and protein expression in transduced cells.
  • Phenylketonuria also called PKU, is a rare inherited disorder that causes an amino acid called phenylalanine to build up in the body. PKU is caused by a change in the phenylalanine hydroxylase (PAH) gene.
  • the methods and systems described herein are used to correct PKU by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the PKU deficiency or integrates a polynucleotide encoding a functional phenylalanine hydroxylase (PAH) gene. Proposed validation is detection of persistent PAH mRNA and protein expression in transduced cells.
  • PAH phenylalanine hydroxylase
  • HCU Homocystinuria
  • Homocystinuria is elevation of the amino acid, homocysteine (protein building block coming from our diet) in the urine or blood.
  • HCU Common causes of HCU include: problems with the enzyme cystathionine beta synthase (CBS), which converts homocysteine to the amino acid cystathionine (which then becomes cysteine) and needs the vitamin B6 (pyridoxine); and problems with converting homocysteine to the amino acid methionine.
  • CBS cystathionine beta synthase
  • pyridoxine pyridoxine
  • problems with converting homocysteine to the amino acid methionine are used to correct HCU by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the HCU or integrates a polynucleotide encoding a functional copy of a gene (e.g., CBS) able to reduce or prevent buildup of homocysteine in the urine.
  • a functional copy of a gene e.g., CBS
  • IgA Nephropathy (Berger’s disease). IgA nephropathy, also known as Berger's disease, is a kidney/autoimmune disease that occurs when an antibody called immunoglobulin A (IgA) builds up in the kidneys.
  • IgA immunoglobulin A
  • the methods and systems described herein are used to treat Berger’s disease by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of Berger’s disease.
  • native cells e.g., B cells
  • the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of Berger’s disease.
  • ANCA vasculitis is an autoimmune disease affecting small blood vessels in the body. It is caused by autoantibodies called ANCAs, or Anti-Neutrophilic Cytoplasmic Autoantibodies. ANCAs target and attack a certain kind of white blood cells called neutrophils.
  • the methods and systems described herein are used to treat ANCA vasculitis by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of ANCA vasculitis.
  • native cells e.g., B cells
  • LN Lupus Nephritis
  • Lupus is an autoimmune—a disorder in which the body’s immune system attacks the body’s own cells and organs.
  • the methods and systems described herein are used to treat SLE/LN by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of SLE/LN.
  • MN Membranous Nephropathy
  • MN is a kidney disease that affects the filters (glomeruli) of the kidney and can cause protein in the urine, as well as decreased kidney function and swelling. It can sometimes be called membranous glomerulopathy as well (these terms can be used interchangeably and mean the same thing).
  • the methods and systems described herein are used to treat MN by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of MN.
  • C3 glomerulonephritis C3GN.
  • C3 glomerulopathy is a group of related conditions that cause the kidneys to malfunction.
  • the major features of C3 glomerulopathy include high levels of protein in the urine (proteinuria), blood in the urine (hematuria), reduced amounts of urine, low levels of protein in the blood, and swelling in many areas of the body. Affected individuals may have particularly low levels of a protein called complement component 3 (or C3) in the blood.
  • the methods and systems described herein are used to treat C3 glomerulopathy by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein.
  • the iPSC-NK cell Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of C3 glomerulopathy. 6.12. Pleiopluripotent Stem Cells [0432] In another embodiment, this disclosure features a pleiopluripotent cell comprising at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome. [0433] As used herein, the term “pleiopluripotent” refers to a pluripotent cell comprising at least a first integration recognition site.
  • Pluripotent refers to the ability of a cell to form all lineages of the body or the embryo proper. Pluripotency is a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell). “Pluripotent” as used herein is intended to include cells that are totipotent, i.e., cells that have the ability to develop into an entire organism.
  • this disclosure features a donor polynucleotide template (e.g., any of the donor polynucleotide templates described herein) comprised of one or more orthogonal integration recognition site integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • a donor polynucleotide template e.g., any of the donor polynucleotide templates described herein
  • the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • At least one of the at least first integration recognition sites is site-specifically incorporated within a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell.
  • at least one of the at least first integration recognition sites is site-specifically incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
  • the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
  • the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus.
  • the locus is the B2M locus.
  • the locus is the CIITA locus.
  • the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
  • the PC-derived cell or population thereof includes at least a first integration recognition site-specifically incorporated into the genome.
  • At least one of the at least first integration recognition sites is specific for a serine integrase. In some embodiments of any of the pleiopluripotent cells described herein, at least one of the at least first integration recognition sites is an attB or attP site. In some embodiments of any of the pleiopluripotent cells described herein, at least one of the at least first integration recognition sites is a modified attB or attP site. In some embodiments of any of the pleiopluripotent cells described herein, at least one of the at least first integration recognition site is specific for BxB1 or a modified BxB1.
  • the pleiopluripotent cell includes a first donor polynucleotide (e.g., any of the donor polynucleotide templates described herein, see, for example, Section 4.11.3) integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, where the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • a first donor polynucleotide e.g., any of the donor polynucleotide templates described herein, see, for example, Section 4.11.3
  • the pleiopluripotent cell includes a second integration recognition site (e.g., any of the integration recognition sites described herein) site-specifically incorporated into the pleiopluripotent cell genome.
  • the second integration recognition sites is site-specifically incorporated into a safe harbor locus.
  • the second integration recognition site is site-specifically incorporated within a second locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell.
  • loci where disruption is capable of reducing allogeneic immune response to the PC-derived cell are as described in Section 4.11.1.
  • the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus.
  • the locus is the B2M locus.
  • the locus is the CIITA locus.
  • the locus where the first integration recognition site is incorporated into the genome and the second integration recognition site is incorporated into the genome are different. In some embodiments, the locus where the first integration recognition site is incorporated into the genome and the second integration recognition site is incorporated into the genome are the same. [0443] In some embodiments of any of the pleiopluripotent cells described herein, the PC-derived cell or population thereof includes a second integration recognition site-specifically incorporated into the genome. In some embodiments of any of the pleiopluripotent cells described herein, the second integration recognition sites is specific for a serine integrase.
  • the second integration recognition sites is an attB or attP site. In some embodiments of any of the pleiopluripotent cells described herein, the second integration recognition sites is a modified attB or attP site. In some embodiments of any of the pleiopluripotent cells described herein, the second integration recognition site is specific for BxB1 or a modified BxB1. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition sites is comprised of 38 or 46 nucleotides.
  • the PC-derived cell or population thereof includes a second donor polynucleotide (e.g., any of the donor polynucleotide templates described herein, see, for example, Section 4.11.3) integrated into the pleiopluripotent stem cell genome at a second integration recognition site, where the second integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • the second donor polynucleotide template encodes one or more therapeutic agents.
  • the one or more therapeutic agents when expressed from the PC-derived cell or population thereof treats, ameliorates, or prevents a disease or condition in a subject.
  • Non-limiting examples of therapeutic agents are as described in Sections 4.11.5 and 4.11.7.
  • the one or more therapeutic agents is a HLA class I proteins.
  • the one or more therapeutic agents is a HLA class I proteins.
  • the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents. In some embodiments of any of the pleiopluripotent cells described herein, the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agent. In some embodiments of any of the pleiopluripotent cells described herein, the second donor polynucleotide template comprises an inducible promoter operably linked to at least one therapeutic agent.
  • the pleiopluripotent cells are derived from a mammal (e.g., a human, rhesus macaque (Macaca mulatta), bovine (Bos taurus), porcine (Sus scrofa), and mouse (Mus musculus)).
  • a mammal e.g., a human, rhesus macaque (Macaca mulatta), bovine (Bos taurus), porcine (Sus scrofa), and mouse (Mus musculus).
  • the pleiopluripotent cells are chimeric (i.e., a combination of iPSC derived from two or species).
  • Non-limiting examples of pleiopluripotent cells include: induced pleiopluripotent cells (iPSC), embryonic stem cells (ESC), somatic cell nuclear transfer embryonic stem cells (ntES cells), epiblast stem cells (EpiSC) and parthenogenesis embryonic stem cell (pES cells).
  • iPSC induced pleiopluripotent cells
  • ESC embryonic stem cells
  • ntES cells somatic cell nuclear transfer embryonic stem cells
  • EpiSC epiblast stem cells
  • pES cells parthenogenesis embryonic stem cell
  • the pleiopluripotent cell is an embryonic stem cell or an induced pleiopluripotent cell.
  • pluripotent cells include cells capable of forming extraembryonic tissue (e.g., cell having totipotency).
  • Pluripotency can be assessed using methods that include, but are not limited to: (i) pleiopluripotent cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pleiopluripotent cell markers (e.g., OCT4, NANOG, SOX2, SSEA3/4, SSEA5, TRA1-60/81, TRAl-85, TRA2-54); (iv) teratoma formation consisting of the three somatic lineages; (v) formation of embryoid bodies consisting of cells from the three somatic lineages ; and (vi) ability to differentiate to all three somatic lineages (ectoderm, mesoderm and endoderm).
  • pleiopluripotent cell markers e.g., OCT4, NANOG, SOX2, SSEA3/4, SSEA5, TRA1-60/81, TRAl-85, TRA2-54
  • teratoma formation consisting of the three somatic lineages
  • embryoid bodies consisting of
  • the pleiopluripotent cell is an induced pluripotent stem cell (iPSC) derived from a non-pluripotent stem cell.
  • iPSC induced pluripotent stem cell
  • the non-pluripotent stem cell can be reprogrammed into an induced pleiopluripotent cell.
  • Non-pluripotent stem cell can be reprogrammed into a induced pluripotent stem cell using exogenous genetic material (e.g., overexpression of transcription factors (e.g., Oct4, Sox2, Klf4, and c-Myc)), exogenous non- genetic material (e.g., small molecules (e.g., (valproic acid (VPA), CHIR99021, 616452, tranylcypromine (VC6T), and Forskolin (FSK (VC6TF))), or a combination thereof.
  • exogenous genetic material e.g., overexpression of transcription factors (e.g., Oct4, Sox2, Klf4, and c-Myc)
  • exogenous non- genetic material e.g., small molecules (e.g., (valproic acid (VPA), CHIR99021, 616452, tranylcypromine (VC6T), and Forskolin (FSK (VC6TF))
  • exogenous genetic material
  • Non-limiting examples of reprogramming a non-pluripotent stem cell into a pluripotent stem cell include methods as described in PCT Publications WO 2022/072883A1, and WO 2022/006399A1; U.S. Patent Nos.10,428,310B2; 9,499,797B2; and 8,802,438B2, each of which are herein incorporated by reference in their entireties.
  • Also provided herein are cell banks comprising any of the pleiopluripotent cells described herein. 6.13.
  • Pleiopluripotent (PC)-derived cell or a population thereof provides a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site (e.g., any of the integration recognition sites described herein) is incorporated site-specifically into the PC- derived cell genome.
  • a first integration recognition site e.g., any of the integration recognition sites described herein
  • this disclosure provides a pleiopluripotent (PC)-derived cell or a population thereof (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • PC pleiopluripotent
  • a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a hematopoietic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site e.g., any of the integration recognition sites described herein) incorporated site-specifically into the PC-derived cell genome.
  • a first integration recognition site e.g., any of the integration recognition sites described herein
  • a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a hematopoietic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • the PC-derived cell is a hematopoietic cell differentiated from a pleiopluripotent cell
  • the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the ple
  • the hematopoietic cell is selected from: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells.
  • HSC hematopoietic stem and progenitor cells
  • MPP hematopoietic multipotent progenitor
  • a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a neuronal cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site (e.g., any of the integration recognition sites described herein) incorporated site-specifically into the PSC-derived cell genome.
  • a first integration recognition site e.g., any of the integration recognition sites described herein
  • a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a neuronal cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • the PC-derived cell is a neuronal cell differentiated from a pleiopluripotent cell
  • the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome
  • the neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron.
  • NPC neural progenitor cell
  • a forebrain neuron a dopaminergic neuron
  • a motor neuron a motor neuron
  • a sensory neuron a sensory neuron.
  • PC-derived Cardiac Cell a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a cardiac cell differentiated from a pleiopluripotent cell; and (ii) the PC- derived cell comprises at least a first integration recognition site (e.g., any of the integration recognition sites described herein) incorporated site-specifically into the PSC-derived cell genome.
  • a first integration recognition site e.g., any of the integration recognition sites described herein
  • a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a cardiac cell differentiated from a pleiopluripotent cell; and (ii) the PC- derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • the cardiac cell is selected from: a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte.
  • a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a pancreatic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site (e.g., any of the integration recognition sites described herein) incorporated site-specifically into the PC-derived cell genome.
  • a first integration recognition site e.g., any of the integration recognition sites described herein
  • a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a pancreatic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • the PC-derived cell is a pancreatic cell differentiated from a pleiopluripotent cell
  • the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome
  • the pancreatic cell is selected from a pancreatic progenitor cell, pancreatic endoderm, an endocrine pancreatic cell, an exocrine pancreatic cell, a islet progenitor, and a beta cell. 6.13.5.
  • the PC-derived cell further comprises two integration recognition sites, three integration recognition sites, four integration recognition sites, five integration recognition sites, six integration recognition sites, seven integration recognition sites, eight integration recognition sites, nine integration recognition sites, or ten or more integration recognition sites.
  • At least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a safe harbor locus.
  • at least one of the at least first integration recognition sites is site-specifically incorporated within a locus, disruption of which is capable of reducing allogeneic immune response to the PC- derived cell.
  • loci where disruption is capable of reducing allogeneic immune response to the PC-derived cell are as described in Section 4.11.1.
  • the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA- C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus.
  • the locus is the B2M locus.
  • the locus is the CIITA locus.
  • the PC-derived cell or population thereof includes at least a first integration recognition site-specifically incorporated into the genome. In some embodiments of any of the PC-derived cell or populations thereof described herein, at least one of the at least first integration recognition sites is specific for a serine integrase. In some embodiments of any of the PC-derived cell or populations thereof described herein, at least one of the at least first integration recognition sites is an attB or attP site. In some embodiments of any of the PC-derived cell or populations thereof described herein, at least one of the at least first integration recognition sites is a modified attB or attP site.
  • At least one of the at least first integration recognition site is specific for BxB1 or a modified BxB1. In some embodiments of any of the PC-derived cell or populations thereof described herein, at least one of the at least first integration recognition sites is comprised of 38 or 46 nucleotides.
  • the PC-derived cell or population thereof includes a first donor polynucleotide (e.g., any of the donor polynucleotide templates described herein, see, for example, Section 4.11.3) integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, where the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. 6.13.6.
  • a first donor polynucleotide e.g., any of the donor polynucleotide templates described herein, see, for example, Section 4.11.3
  • the PC-derived cell or population thereof includes a second integration recognition site (e.g., any of the integration recognition sites described herein) site-specifically incorporated into the pleiopluripotent cell genome.
  • the second integration recognition sites is site-specifically incorporated into a safe harbor locus.
  • the second integration recognition site is site-specifically incorporated within a second locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell.
  • loci where disruption is capable of reducing allogeneic immune response to the PC- derived cell are as described in Section 4.11.1.
  • the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus.
  • the locus is the B2M locus.
  • the locus is the CIITA locus.
  • the locus where the first integration recognition site is incorporated into the genome and the second integration recognition site is incorporated into the genome are different. In some embodiments, the locus where the first integration recognition site is incorporated into the genome and the second integration recognition site is incorporated into the genome are the same. [0473] In some embodiments of any of the PC-derived cell or populations thereof described herein, the PC-derived cell or population thereof includes a second integration recognition site- specifically incorporated into the genome. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition sites is specific for a serine integrase. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition sites is an attB or attP site.
  • the second integration recognition sites is a modified attB or attP site. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition site is specific for BxB1 or a modified BxB1. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition sites is comprised of 38 or 46 nucleotides.
  • the PC-derived cell or population thereof includes a second donor polynucleotide (e.g., any of the donor polynucleotide templates described herein, see, for example, Section 4.11.3) integrated into the pleiopluripotent stem cell genome at a second integration recognition site, where the second integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
  • the second donor polynucleotide template encodes one or more therapeutic agents.
  • the one or more therapeutic agents when expressed from the PC-derived cell or population thereof treats, ameliorates, or prevents a disease or condition in a subject.
  • therapeutic agents are as described in Sections 4.11.5and 4.11.7.
  • the one or more therapeutic agents is a HLA class I proteins.
  • the one or more therapeutic agents is a HLA class I proteins.
  • the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents.
  • the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agent. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second donor polynucleotide template comprises an inducible promoter operably linked to at least one therapeutic agent. 6.14.
  • Kits and Cell-based Compositions [0477] This disclosure also provides certain components or embodiments of cell-based products (e.g., genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell) or pharmaceutical compositions thereof, can be provided in a kit.
  • cell-based products e.g., genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell
  • pharmaceutical compositions thereof can be provided in a kit.
  • any of the cell-based products described herein e.g., any of the genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a pancreatic cell
  • any of the cell-based products described herein can be provided frozen and packaged as a kit, alone or along with separate containers of any of the other agents from the pre- conditioning or post-conditioning steps, and optional instructions for use.
  • the cell-based products e.g., genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell
  • the cell-based products may be frozen (cryopreserved) before administration into a subject.
  • cell-based products e.g., genetically modified pleiopluripotent cells or cells- derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell
  • cell-based products may be frozen by suspending the cells in media containing at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 50%) human-derived serum and/or plasma, and lowering the temperature of the suspension to at least -80° C, thereby freezing the cell-based products.
  • the freezing media is approximately 30% human- derived serum and/or plasma and approximately 10% of an agent that prevents ice crystal formation during freezing, e.g., DMSO.
  • the cell-based product e.g., genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell
  • freezing media suspension is maintained at -80°C for at least 24 hours and then transferred to liquid nitrogen for the duration of the storage.
  • the frozen cell-based product e.g., genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell
  • the frozen cell-based product e.g., genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell
  • the frozen cell-based product e.g., genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell
  • the genetically modified pleiopluripotent cells are selected, isolated, or a combination thereof, prior to being banked (e.g., cryopreserved).
  • the genetically modified pleiopluripotent cells are selected in bulk before being bank
  • the genetically modified pleiopluripotent cells are isolated prior to being banked (e.g., cryopreserved). In such cases, the genetically modified pleiopluripotent cells are isolated and expanded prior to being banked (e.g., cryopreserved).
  • the genetically modified pluripotent stem cells are banked (e.g., cryopreserved) following incorporation of one or more integration recognition sites (e.g., any of the integration recognition sites described herein) but prior to integration of a donor polynucleotide template (e.g., any of the donor polynucleotide templates described herein).
  • the genetically modified pluripotent stem cells are thawed (and optionally allowed to recover) prior to integrating a donor polynucleotide template (e.g., any of the donor polynucleotide templates described herein) into the genome of the pluripotent stem cell.
  • a donor polynucleotide template e.g., any of the donor polynucleotide templates described herein
  • the genetically modified pluripotent stem cells are banked (e.g., cryopreserved) following incorporation of one or more integration recognition sites (“beacons”) (e.g., any of the integration recognition sites described herein) and integration of a donor polynucleotide template (e.g., any of the donor polynucleotide templates described herein).
  • beacons integration recognition sites
  • the genetically modified pluripotent stem cells can be thawed (and optionally allowed to recover) prior to integrating a second donor polynucleotide template (e.g., any of the donor polynucleotide templates described herein) into the genome of the pluripotent stem cell.
  • a second donor polynucleotide template e.g., any of the donor polynucleotide templates described herein
  • the genetically modified pluripotent stem cells can be thawed (and optionally allowed to recover) and used for downstream applications (e.g., differentiation to a hematopoietic stem cell, a neuronal cell, a cardiac cell, or a beta cell and use as a source for cell therapy).
  • the cells are frozen, which can be referred to as cryopreservation.
  • the cells e.g., genetically modified pluripotent stem cells
  • the cells can, for example, be frozen using CryoStor CS5 (freeze media), Cryostor CS10 (freeze media), or another freeze media.
  • CryoStor CS5 freeze media
  • Cryostor CS10 freeze media
  • another freeze media this is a non-limiting example of freezing media and other freezing media can be used.
  • the cells e.g., genetically modified pluripotent stem cells
  • the cells can be initially chilled 4°C. Then the cells (e.g., genetically modified pluripotent stem cells) can be further cooled to about -20°C.
  • the cells can be centrifuged or resuspended in additional freeze media and then cooled to -90°C in a stepwise manner.
  • the cells e.g., genetically modified pluripotent stem cells
  • the cells can then be stored in liquid nitrogen.
  • the cells e.g., genetically modified pluripotent stem cells
  • the composition may comprise a pharmaceutically acceptable excipient, a pharmaceutically acceptable salt, diluents, carriers, vehicles and such other inactive agents well known to the skilled artisan.
  • Vehicles and excipients commonly employed in pharmaceutical preparations include, for example, talc, gum Arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, etc. Solutions can be prepared using water or physiologically compatible organic solvents such as ethanol, 1,2-propylene glycol, polyglycols, dimethylsulfoxide, fatty alcohols, triglycerides, partial esters of glycerine and the like.
  • Compositions may be prepared using conventional techniques that may include sterile isotonic saline, water, 1,3-butanediol, ethanol, 1,2-propylene glycol, polyglycols mixed with water, Ringer's solution, etc.
  • a coloring agent is added to facilitate in locating and properly placing the composition to the intended treatment site.
  • Compositions may include a preservative and/or a stabilizer.
  • Non-limiting examples of preservatives include methyl-, ethyl-, propyl-parabens, sodium benzoate, benzoic acid, sorbic acid, potassium sorbate, propionic acid, benzalkonium chloride, benzyl alcohol, thimerosal, phenylmercurate salts, chlorhexidine, phenol, 3-cresol, quaternary ammonium compounds (QACs), chlorbutanol, 2-ethoxyethanol, and imidurea.
  • the composition can comprise a physiological salt, such as a sodium salt.
  • Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml.
  • compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included at a concentration in the 5-20 mM range. The pH of a composition will generally be between 5 and 8, and more typically between 6 and 8 e.g. between 6.5 and 7.5, or between 7.0 and 7.8. [0487] In some embodiments, the composition may include a cryoprotectant agent.
  • cryoprotectant agents include a glycol (e.g., ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, and any combinations thereof.
  • a glycol e.g., ethylene glycol, propylene glycol, and glycerol
  • DMSO dimethyl sulfoxide
  • the kit may comprise ampoules, disposable syringes, capsules, vials, tubes, or the like.
  • the kit may comprise a single dose container or multiple dose containers comprising the topical formulation of embodiments herein.
  • each dose container may contain one or more unit doses.
  • the kit may include an applicator.
  • kits include all components needed for the stages of conditioning/treatment.
  • the cellular compositions may have preservatives or be preservative-free (for example, in a single-use container).
  • the cell-based products may be prepared and frozen at a desired stage, suitable for shipping to a hospital or treatment center.
  • Pharmaceutical Compositions [0490] This disclosure provides pharmaceutical compositions comprising the pluripotent stem cell or hematopoietic cell, cardiac cell or neuronal cell derived from the pluripotent stem cell, wherein the pharmaceutical compositions further comprise a pharmaceutically acceptable medium.
  • the pharmaceutical composition comprises the pluripotent cell derived hematopoietic cells made by the methods and composition disclosed herein. In one embodiment, the pharmaceutical composition comprises the pluripotent cell derived cardiac cells made by the methods and composition disclosed herein. In one embodiment, the pharmaceutical composition comprises the pluripotent cell derived neuronal cells made by the methods and composition disclosed herein. 6.16. Directed Differentiation of Pleiopluripotent Cells [0491] In certain embodiments, the method further includes directing differentiation of the modified pluripotent stem cell to a hematopoietic cell.
  • Non-limiting examples of a hematopoietic cell include: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, platelets, erythrocytes, erythrocyte progenitors, megakaryocytes, and megakaryocyte precursors.
  • HSC hematopoietic stem and progenitor cells
  • MPP multipotent progenitor
  • the method further includes directing differentiation of the modified pluripotent stem cell to a neuronal cell.
  • neuronal cells include: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron.
  • Non-limiting methods of directing differentiation of the modified pluripotent stem cell to a neuronal cell include the methods described in the PCT Publication Nos. WO2022/134031A1; WO2022/136306A1; WO2022/093375A1; WO2022/014681A1; and WO2022005023A1; U.S. Patent Nos. 11,007,232; 10,519,421; and 10,487,311; and U.S. Patent Pubs.2021/0244768A1, each of which are herein incorporated by reference in their entireties. [0493] In certain embodiments, the method further comprises directing differentiation of the modified pluripotent stem cell to a cardiac cell.
  • Non-limiting examples of a cardiac cell include: a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte.
  • Non-limiting methods of directing differentiation of the modified pluripotent stem cell to a cardiomyocyte include the methods described in PCT Publication Nos. WO2020/247957; WO2021/187601A1; WO2021/172542A1; and WO2020/264308A1; and U.S. Patent Nos. 11,352,604; 10,973,876; 10,443,044; and 9,587,220; and U.S. Patent Pubs.2021/0363487A1, and 2020/0140819A1, each of which are herein incorporated by reference in their entireties.
  • the method further comprises directing differentiation of the modified pluripotent stem cell to a pancreatic cell.
  • a pancreatic cell include: a pancreatic progenitor cell, pancreatic endoderm, an endocrine pancreatic cell, an exocrine pancreatic cell, a islet progenitor, and a beta cell.
  • Non-limiting methods of directing differentiation of the modified pluripotent stem cell to a beta cell include the methods described in PCT Publication Nos. WO2020/033879A1; WO2021/030424A1; and WO2020/264072A1, U.S. Patent Nos. 10,376,545; and 9,650,610; U.S. Patent Publication No.
  • This disclosure also provides a method of using a pleiopluripotent cell having at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome, the method comprising: integrating a first donor polynucleotide template into the pleiopluripotent cell genome by introducing into the pleiopluripotent cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the at least one incorporated genomic integration recognition sites by the integrase.
  • the method of using pleiopluripotent cells also includes selecting the pleiopluripotent stem cells having the first donor polynucleotide template site-specifically integrated into the genome. In some embodiments, the method of using pleiopluripotent cells also includes expanding the pleiopluripotent cells in a de-differentiated state. [0497] In some embodiments, the method of using pleiopluripotent cells also includes cryopreserving the pleiopluripotent cells following incorporation of the at least first integration recognition site.
  • the method of using pleiopluripotent cells also includes cryopreserving the pleiopluripotent cells following integrating the donor polynucleotide template at the at least one incorporated genomic recognition site. [0498] In some embodiments, the method of using the pleiopluripotent cells also includes directing differentiation of the modified pleiopluripotent cell to a hematopoietic cell.
  • Non- limiting examples of hematopoietic cells include: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells.
  • the method of using the pleiopluripotent cells also includes directing differentiation of the modified pleiopluripotent cell to a neuronal cell.
  • Non-limiting examples of a neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron.
  • NPC neural progenitor cell
  • the method of using the pleiopluripotent cells also includes directing differentiation of the modified pleiopluripotent cell to a cardiac cell.
  • cardiac cells include a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte.
  • the method of using the pleiopluripotent cells also includes directing differentiation of the modified pleiopluripotent cell to a pancreatic cell.
  • pancreatic cells include pancreatic progenitor cell, pancreatic endoderm, an endocrine pancreatic cell, an exocrine pancreatic cell, an islet progenitor, and a beta cell.
  • the method of using the pleiopluripotent cells also includes administering the PC-derived cells or a population thereof to a subject (patient) in need thereof. 6.18. Programmable Gene Insertion for Research Purposes [0503]
  • the methods and compositions described herein can be used to generate cell lines for research purposes or applications that are intended for research purposes only.
  • the methods can be used for research purposes or applications that are intended for research purposes only.
  • the methods can be used for screening purposes, for example, screening therapeutic molecules (e.g., compounds, small molecules, and large molecules). 6.19. Methods of Treatment [0504] In another aspect, methods of treatment are presented.
  • the subject has a disease, condition, and/or an injury that can be treated, ameliorated, and/or improved by a cell therapy.
  • a subject in need of cell therapy is a subject with an injury, disease, or condition, whereby a cell therapy, for example, a therapy in which a cellular material is administered to the subject, can treat, ameliorate, improve, and/or reduce the severity of at least one symptom associated with the injury, disease, or condition.
  • a subject in need of cell therapy includes, but is not limited to, a candidate for bone marrow or stem cell transplantation, a subject who has received chemotherapy or irradiation therapy, a subject who has or is at risk of having a hyperproliferative disorder or a cancer, (e.g., a hyperproliferative disorder) or a cancer of hematopoietic system, a subject having or at risk of developing a tumor (e.g., a solid tumor), a subject who has or is at risk of having a viral infection or a disease associated with a viral infection.
  • a hyperproliferative disorder or a cancer e.g., a hyperproliferative disorder
  • a cancer of hematopoietic system e.g., a cancer of hematopoietic system
  • a subject having or at risk of developing a tumor e.g., a solid tumor
  • This disclosure also features a method of treating or ameliorating or preventing a disease or condition in a subject, comprising administering a therapeutically effective amount of any of the PC-derived cell or population thereof, compositions, or pharmaceutical composition described herein.
  • the method includes treating or ameliorating or preventing cancer, where the subject has, or is suspected of having, cancer.
  • the method includes treating or ameliorating or preventing a cardiac condition, where the subject has or is suspected of having, a cardiac condition. In such cases, the cardiac condition is muscular and/or the condition is muscle degeneration or muscle injury.
  • the method includes treating or ameliorating or preventing a neuronal-associated disease, wherein the subject has, or is suspected of having, a neuronal- associated disease. In such cases, the disease is a neuronal-associated disease and/or the condition is neuron degeneration. [0510] In some embodiments, the method includes treating or ameliorating or preventing a pancreatic-associated disease, wherein the subject has, or is suspected of having, a pancreatic- associated disease. [0511] In some embodiments, the method of treating further includes administering to the subject, having previously been administered any of pluripotent stem cells described herein, one or more exogenous molecules to control activity of the one or more inducible suicide genes.
  • the method of treating also includes, following administration of any of the PC- derived cells or populations thereof as described herein, administering AP20187 (or an analog thereof), AP21967, ganciclovir, or a combination thereof.
  • the method of cell delivery used here occurs using electroporation.
  • the method of cell delivery used here occurs using a recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, anellovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, mRNA, RNP, or lipid nanoparticle.
  • nucleic acid construct can also be by fusosome or exosome, (See, e.g., WO2019222403 which is incorporated by reference herein). Delivery of nucleic acid construct can also be by VesiCas (See, e.g., US20210261957A1 which is incorporated by reference herein).
  • Methods of non-viral delivery include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos.
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). 6.19.1. Viral Vector Delivery into the Pluripotent Stem Cell Genome [0515] In certain aspects the invention involves vectors, e.g.
  • a "vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double- stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g.
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors.” Vectors for and that result in expression in a eukaryotic cell can be referred to herein as “eukaryotic expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • Vector delivery e.g., plasmid, viral delivery:
  • the CRISPR enzyme for instance a Type V protein such as C2c1 or C2c3, and/or any of the present RNAs, for instance a guide RNA, can be delivered using any suitable vector, e.g., plasmid or viral vectors, such as adeno associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof.
  • AAV adeno associated virus
  • effector proteins and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmid or viral vectors.
  • the promoter used to drive nucleic acid-targeting effector protein coding nucleic acid molecule expression can include: AAV ITR can serve as a promoter: this is advantageous for eliminating the need for an additional promoter element (which can take up space in the vector). The additional space freed up can be used to drive the expression of additional elements (gRNA, etc.). Also, ITR activity is relatively weaker, so can be used to reduce potential toxicity due to over expression of nucleic acid-targeting effector protein.
  • promoters CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc.
  • promoters For brain or other CNS expression, can use promoters: Synapsin I for all neurons, CaMKII alpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.
  • For liver expression can use Albumin promoter.
  • For lung expression can use SP-B.
  • endothelial cells can use ICAM.
  • hematopoietic cells can use IFNbeta or CD45.
  • Osteoblasts can use OG-2.
  • the promoter used to drive guide RNA can include: Pol III promoters such as U6 or H1 Use of Pol II promoter and intronic cassettes to express guide RNA Adeno Associated Virus (AAV) [0520] Nucleic acid-targeting effector protein and one or more guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Pat. No.8,454,972 (formulations, doses for adenovirus), U.S. Pat. No.8,404,658 (formulations, doses for AAV) and U.S. Pat. No.
  • 5,846,946 formulations, doses for DNA plasmids
  • the route of administration, formulation and dose can be as in U.S. Pat. No. 8,454,972 and as in clinical trials involving AAV.
  • the route of administration, formulation and dose can be as in U.S. Pat. No. 8,404,658 and as in clinical trials involving adenovirus.
  • the route of administration, formulation and dose can be as in U.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.
  • Doses may be based on or extrapolated to an average 70 kg individual (e.g., a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed.
  • the viral vectors can be injected into the tissue of interest.
  • the expression of nucleic acid-targeting effector can be driven by a cell-type specific promoter.
  • liver-specific expression might use the Albumin promoter and neuron-specific expression (e.g., for targeting CNS disorders) might use the Synapsin I promoter.
  • mRNA delivery methods and compositions that may be utilized in the present disclosure including, for example, PCT/US2014/028330, US8822663B2, NZ700688A, ES2740248T3, EP2755693A4, EP2755986A4, WO2014152940A1, EP3450553B1, BR112016030852A2, and EP3362461A1.
  • Expression of CRISPR systems in particular is described by WO2020014577.
  • Each of these publications are incorporated herein by reference in their entireties.
  • one or more vectors described herein are used to produce a non- human transgenic animal or transgenic plant.
  • the transgenic animal is a mammal, such as a mouse, rat, or rabbit.
  • the organism or subject is a plant.
  • the organism or subject or plant is algae. Methods for producing transgenic plants and animals are known in the art, and generally begin with a method of cell transfection, such as described herein.
  • the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including micro-algae) and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant (including micro-algae).
  • target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide.
  • target polynucleotides include a disease associated gene or polynucleotide.
  • a “disease-associated" gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease.
  • a disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.
  • the transcribed or translated products may be known or unknown and may be at a normal or abnormal level.
  • the genetically modified cell are non-dividing cells (e.g., where cell growth has been arrested) prior to being contacted with the gene editing reagents (e.g., gene editor protein or a polynucleotide encoding a gene editor protein, one or more guide RNA, and optionally a nicking gRNA).
  • the gene editing reagents e.g., gene editor protein or a polynucleotide encoding a gene editor protein, one or more guide RNA, and optionally a nicking gRNA.
  • the genetically modified cells are terminally differentiated, non-dividing cells prior to being contacted with the gene editing reagents (e.g., gene editor protein or a polynucleotide encoding a gene editor protein, one or more guide RNA, and optionally a nicking gRNA). In some embodiments, the genetically modified cells are dividing cells. 7. ADDITIONAL EMBODIMENTS [0526] Embodiment 1.
  • a method of editing a pluripotent stem cell comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the one or more integration target recognition sites into the genome; and (ii) two guide RNAs, wherein each of the two guide RNA encodes all or part of an integrase target recognition site.
  • a method of editing a pluripotent stem cell comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the one or more integration target recognition sites into the genome; and (ii) two guide RNA complexes, wherein each of the two guide RNA complexes is comprised of at least one polynucleotide that encodes all or part of an integrase target recognition site.
  • a method of editing a pluripotent stem cell comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the one or more integration target recognition sites into the genome; (ii) one or more guide RNA, wherein the one or more guide RNA encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA.
  • a method of editing a pluripotent stem cell comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of polynucleotide genomic integration; (ii) one or more guide RNA complex, wherein the one or more guide RNA complex is comprised of at least one polynucleotide that encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA. [0530] Embodiment 5.
  • any one of embodiments 1-4 further comprising: b) integrating a donor polynucleotide template into the pluripotent stem cell genome at the one or more incorporated target recognition sites by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration target recognition sites, wherein the donor polynucleotide is integrated into the pluripotent stem cell at the one or more incorporated genomic integration target recognition sites by an integrase; thereby producing a genetically modified pluripotent stem cell.
  • Embodiment 6 Embodiment 6.
  • a method of editing a pluripotent stem cell comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the one or more integration target recognition sites into the genome; and (ii) two guide RNAs, wherein each of the two guide RNA encodes all or a portion of an integrase target recognition site; and (b) integrating a donor polynucleotide template into the pluripotent stem cell genome at the one or more incorporated target recognition sites by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration target recognition sites, wherein the donor polynucleotide is integrated into the pluripotent stem cell at the incorporated genomic integration target recognition site by an integrase; thereby
  • Embodiment 7 A method of editing a pluripotent stem cell genome, the method comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of polynucleotide genomic integration; and (ii) two guide RNA complexes, wherein each of the two guide RNA complexes is comprised of at least one polynucleotide that encodes all or part of an integrase target recognition site; and (b) integrating a donor polynucleotide template into the pluripotent stem cell genome at the one or more incorporated target recognition sites by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one of more integration target recognition sites, wherein the donor polynucleotide is integrated into the pluripot
  • Embodiment 8 A method of editing a pluripotent stem cell, the method comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the one or more integration target recognition sites into the genome; (ii) one or more guide RNA, wherein the one or more guide RNA encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA; and (b) integrating a donor polynucleotide template into the pluripotent stem cell genome at the one or more incorporated target recognition sites by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration target recognition sites, wherein the donor polynucleotide is integrated into the pluripotent
  • Embodiment 9 A method of editing a pluripotent stem cell genome, the method comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of polynucleotide genomic integration; (ii) one or more guide RNA complex, wherein the one or more guide RNA complex is comprised of at least one polynucleotide that encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA; and (b) integrating a donor polynucleotide template into the pluripotent stem cell genome at the one or more incorporated target recognition sites by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one of more integration target recognition sites, wherein the donor polynu
  • Embodiment 10 The method of any one of embodiments 6-9, wherein steps (a) and (b) are performed concurrently.
  • Embodiment 11 The method of any one of embodiments 1-10, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
  • Embodiment 12 The method of any one of embodiments 1-11, wherein the one or more integration target recognition sites is incorporated at one or more loci associated with HLA Class I proteins, one or more loci associated with HLA Class II proteins, or a combination thereof.
  • Embodiment 13 Embodiment 13.
  • the pluripotent stem cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
  • Embodiment 15 The method of embodiment 14, wherein the one or more HLA Class I proteins, one or more HLA Class II proteins are encoded in one or more chromosomal regions selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
  • Embodiment 16 The method of embodiment 14, wherein the one or more HLA Class I proteins or one or more HLA Class II proteins are selected from: B2M, and CIITA.
  • Embodiment 18 The method of any one of embodiments 1-16, wherein delivery is performed by electroporation.
  • Embodiment 18 The method of any one of embodiments 1-17, wherein delivery into the pluripotent stem cell occurs using one or more of a recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, anellovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, mRNA, RNP, or lipid nanoparticle.
  • Embodiment 19 The metho of any one of embodiments 1-16, wherein delivery is performed by transfection.
  • Embodiment 20 The metho of any one of embodiments 1-16, wherein delivery is performed by transfection.
  • Embodiment 21 The method of any one of embodiments 1-20, wherein the one or more integration target recognition sites is specific for a serine integrase.
  • Embodiment 22 The method of any one of embodiments 1-21, wherein the one or more integration target recognition sites is an attB or attP site.
  • Embodiment 23 The method of any one of embodiments 1-21, wherein the one or more integration target recognition sites is an attB or attP site.
  • Embodiment 24 The method of any one of embodiments 1-23, wherein the one or more integration target recognition sites is specific for BxB1 or a modified BxB1.
  • Embodiment 25 The method of any one of embodiments 1-24, wherein the one or more integration target recognition sites is comprised of 38 or 46 nucleotides.
  • Embodiment 26 The method of any one of embodiments 1-25, wherein the one or more guide RNA or the one or more guide RNA complex comprises a chemical modification.
  • Embodiment 27 Embodiment 27.
  • Embodiment 28 The method of embodiment 27, wherein expression of the one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof, is driven by a pluripotent stem cell-specific promoter.
  • Embodiment 29 The method of embodiment 27, wherein expression of the one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof, is driven by a constitutive promoter.
  • Embodiment 30 The method of any one of embodiments 27-29, wherein the one or more inducible suicide genes is selected from: caspase9, cytosine deasminase, and thymidine kinase.
  • Embodiment 31 The method of embodiment 30, wherein the one or more inducible suicide genes is a controllable caspase9.
  • Embodiment 32 The method of embodiment 31, wherein AP20187 (or an analog thereof) controls activity of Caspase9 or AP21967 (or an analog thereof) controls activity of Caspase9.
  • Embodiment 33 Embodiment 33.
  • Embodiment 34 The method of embodiment 33, wherein the polynucleotide encoding for the thymidine kinase is operably linked to the controllable capase9 protein using a 2A self-cleaving peptide or an internal ribosome entry site.
  • Embodiment 35 Embodiment 35.
  • any one of embodiments 1-34, wherein the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL- 1, B2M-HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof.
  • Embodiment 36 The method of embodiment 35, wherein the donor polynucleotide template encodes a CD47, PDL1, and B2M-HLA-E.
  • Embodiment 37 Embodiment 37.
  • Embodiment 38 The method of any one of embodiments 27-37, wherein the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag.
  • Embodiment 39 The method of any one of embodiments 27-38, wherein the donor polynucleotide template that encodes one or more inducible suicide genes further comprises an integrase target recognition site.
  • Embodiment 40 The method of any one of embodiments 27-38, wherein the donor polynucleotide template that encodes one or more inducible suicide genes further comprises an integrase target recognition site.
  • any one of embodiments 1-40 further comprising: incorporating an additional integration target recognition site into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of polynucleotide genomic integration; (ii) one or more guide RNA or one or more guide RNA complex, wherein the one or more guide RNA or one or more guide RNA complex is comprised of at least one polynucleotide that encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA. [0567] Embodiment 42.
  • the method of embodiment 41 further comprising: integrating a second donor polynucleotide template into the pluripotent stem cell genome at the incorporated additional target recognition site by delivering into the cell: (i) the second donor polynucleotide template, wherein the second donor polynucleotide template is comprised of an additional integration target recognition site, wherein the second donor polynucleotide is integrated into the pluripotent stem cell at the incorporated additional genomic integration target recognition site by an integrase; thereby producing a genetically modified pluripotent stem cell comprising a first polynucleotide template and a second polynucleotide template each integrated into the pluripotent stem cell genome.
  • Embodiment 43 Embodiment 43.
  • Embodiment 44 The method of embodiment 43, wherein the hematopoietic cell is selected from: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells.
  • HSC hematopoietic stem and progenitor cells
  • MPP hematopoietic multipotent progenitor
  • Embodiment 46 The method of embodiments 45, wherein the neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron.
  • NPC neural progenitor cell
  • Embodiment 47 The method of any one of embodiments 1-42, further comprising directing differentiation of the modified pluripotent stem cell to a cardiac cell.
  • Embodiment 48 Embodiment 48.
  • a pluripotent stem cell comprising: (a) an integration target recognition site integrated in the pluripotent stem cell genome at one or more loci associated with HLA Class I proteins, one or more loci associated with HLA Class II proteins, or a combination thereof.
  • Embodiment 50 A pluripotent stem cell comprising: (a) a donor polynucleotide template integrated into the pluripotent stem cell genome at one or more loci associated with HLA Class I proteins, one or more loci associated with HLA Class II proteins, or a combination thereof.
  • a pluripotent stem cell comprising: (a) an integration target recognition site that is integrated (or capable of being integrated) in the pluripotent stem cell genome at one or more loci associated with HLA Class I proteins, one or more loci associated with HLA Class II proteins, or a combination thereof; (b) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the integration target recognition site into the genome; (c) one or more guide RNA, wherein the one or more guide RNA encodes an integrase target recognition site; and (d) optionally, a nicking gRNA.
  • Embodiment 52 Embodiment 52.
  • the pluripotent stem cell of embodiment 51 further comprising: (e) a donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration target recognition site, wherein the donor polynucleotide is integrated into the pluripotent stem cell genome at the incorporated genomic integration target recognition site.
  • Embodiment 53 The pluripotent stem cell of any one of embodiments 49-52, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
  • Embodiment 54 Embodiment 54.
  • Embodiment 55 Embodiment 55.
  • Embodiment 56 Embodiment 56.
  • Embodiment 57 The pluripotent stem cell of embodiment 56, wherein the one or more HLA Class I proteins or one or more HLA Class II proteins are selected from: B2M and CIITA.
  • Embodiment 58 The pluripotent stem cell of any one of embodiments 49-57, wherein the integration target recognition site is an attB or attP site.
  • Embodiment 59 Embodiment 59.
  • Embodiment 60 The pluripotent stem cell of any one of embodiments 49-59, wherein the integration target recognition site is specific for BxB1 or a modified BxB1.
  • Embodiment 61 The pluripotent stem cell of any one of embodiments 49-60, wherein the integration target recognition site is comprised of 38 or 46 nucleotides.
  • Embodiment 62 The pluripotent stem cell of any one of embodiments 49-61, wherein the one or more guide RNA or the one or more guide RNA complex comprises a chemical modification.
  • Embodiment 63 The pluripotent stem cell of any one of embodiments 49-62, wherein the donor polynucleotide template encodes for one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof.
  • Embodiment 64 The pluripotent stem cell of embodiment 63, wherein expression of the one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof, is driven by a pluripotent stem cell-specific promoter.
  • Embodiment 65 Embodiment 65.
  • Embodiment 66 The pluripotent stem cell of any one of embodiments 63-65, wherein the one or more inducible suicide genes is select from: caspase9, cytosine deasminase, and thymidine kinase.
  • Embodiment 67 The pluripotent stem cell of embodiment 66, wherein the one or more inducible suicide genes is a controllable caspase9.
  • Embodiment 69 The pluripotent stem cell of embodiment 67, wherein AP20187 (or analog thereof) controls activity of Caspase9 or AP21967 (or analog thereof) controls activity of Caspase9.
  • Embodiment 69 The pluripotent stem cell of embodiment 67, further comprising a second inducible suicide gene, wherein the second inducible suicide gene comprises a thymidine kinase.
  • Embodiment 70 Embodiment 70.
  • Embodiment 71 The pluripotent stem cell of any one of embodiments 63-70, wherein the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL-1, B2M-HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof.
  • Embodiment 72 Embodiment 72.
  • the pluripotent stem cell of embodiment 71 wherein the donor polynucleotide template encodes a CD47, PDL1, and B2M-HLA-E.
  • Embodiment 73 The pluripotent stem cell of embodiment 72, wherein the polynucleotide template encoding CD47, PDL1 and B2M-HLA-E are separated by a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof.
  • Embodiment 74 The pluripotent stem cell of any one of embodiments 63-73, wherein the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag.
  • Embodiment 75 The pluripotent stem cell of embodiments 63-71, wherein the one or more inducible suicide genes further comprise an integrase target recognition site.
  • Embodiment 76 The pluripotent stem cell of any one of embodiments 63-75, wherein the one or more exogenous polypeptides capable of modulating an immune response further comprises an integrase target recognition site.
  • Embodiment 77 A composition comprising the pluripotent stem cell of embodiments 49- 76.
  • a pharmaceutical composition one or more of the pluripotent stem cells of claims 49-76 and a pharmaceutically acceptable excipient. 8. EXAMPLES 8.1.
  • the dual atgRNAs each included a portion of the 38 bp attB insertion site within a segment of the reverse transcriptase template (see, e.g., FIGs.7A-7B) (Table 12).
  • the protospacer sequences i.e., a sequence/domain capable of guiding the gene editor protein or prime editor fusion protein to site-specifically nick the iPSC genome
  • FIG.8 A non-limiting example of a method of generating an iPSC having an integration recognition site site-specifically integrated into the iPSC genome is shown in FIG.8.
  • iPSC lines ACS-1030 (iPSC1) and ACS-1026 (iPSC2) were purchased from ATCC and cultured as undifferentiated iPSCs grown on Cell Basement Membrane (ATCC)-coated plates in Pluripotent Stem Cell SFM XF/FF media (ATCC).
  • Undifferentiated iPSCs were electroporated with prime editor fusion protein (2 micrograms mRNA encoding prime editor fusion protein) and atgRNA (50 picomoles). Electroporation was performed using a Neon Transfection System (ThermoFisher) with a Neon P10 tip at 1200V, 30 msec, 1 pulse.
  • FIGs. 1-10 Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane-coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 ⁇ M Rho Kinase (ROCK) inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, media was changed and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and beacon placement at the B2M locus and/or the CIITA locus was measured using ddPCR and/or AmpSeq. [0607] FIGs.
  • FIGS 10A-10B show beacon placement in iPSC lines ACS-1030 (iPSC1) and ACS- 1026 (iPSC2) at B2M exon 1 and/or CIITA exon 2 for each iPSC line.
  • FIG.10A shows beacon placement in iPSC line 1 (ACS-1030) at the B2M locus was between about 15% to about 95% as measured by ddPCR and between about 30% to about 95% as measured by AmpSeq.
  • FIG.10A shows beacon placement in iPSC line 1 (ACS-1030) at the CIITA locus was between about 25% to about 45% as measured by ddPCR and between about 30% to about 55% as measured by AmpSeq.
  • FIG.10B shows beacon placement in iPSC line 2 (ACS-1026) at the B2M locus was between about 10% to about 75% as measured by ddPCR.
  • FIG. 10B also shows beacon placement in iPSC line 2 (ACS-1026) at the CIITA locus was between about 10% to about 15% as measured by ddPCR.
  • Example 2 Concurrent beacon placement at B2M and CIITA loci in iPSCs using a dual atgRNA approach at each locus
  • a dual atgRNA approach to beacon placement was used for beacon placement at each locus.
  • the same dual atgRNAs as described in Example 1 were used in this Example.
  • a non-limiting example of a method of generating an iPSC where the method includes concurrent site-specific integration of integration recognition sites in the B2M locus and CIITA locus is shown in FIG.8.
  • iPSC lines ACS-1030 (iPSC1) and ACS-1026 (iPSC2) were cultured as undifferentiated iPSCs on Cell Basement Membrane (ATCC)-coated plates in Pluripotent Stem Cell SFM XF/FF media (ATCC).
  • Undifferentiated iPSCs were electroporated with prime editor fusion protein (2 micrograms mRNA encoding prime editor fusion protein) and atgRNA (50 picomoles). Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse.
  • FIGs.11A-11B shows concurrent beacon placement at B2M and CIITA loci using the dual atgRNA approach for beacon placement at each locus as described above.
  • FIG.11A shows ddPCR plots, with the top panel showing data for target specific probes and the bottom panel showing data for control probes.
  • FIG. 11B shows a summary histogram from the ddPCR data presented in the FIG.11A.
  • FIGs. 11A-11B show successful concurrent beacon placement at B2M and CIITA loci with at least 84.9% beacon placement for B2M and at least 51.3% beacon placement for CIITA.
  • electroporated iPSCs were plated on Cell Basement Membrane-coated plates in Pluripotent Stem Cell SFM XF/FF media and allowed to recover.
  • iPSCs were plated at a density suitable to allow the single cells to grow into iPSC colonies that could be picked and expanded for purposes of cell line generation.
  • iPSCs were grown in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 ⁇ M ROCK inhibitor Y27632.
  • colonies were picked, disassociated into clumps (e.g., no less than 4 cells per clump) and re-plated onto a Cell Basement Membrane-coated dish.
  • the picked iPSC lines were expanded, analyzed for beacon placement, and cryopreserved.
  • FIG. 12A shows raw ddPCR data for beacon placement at the B2M locus and the CIITA locus for 22 iPSC lines.
  • FIG. 12B is a histogram summarizing ddPCR data for three iPSC lines: line 1, line 8, and line 9. Line 1 and line 9 show 100% bi-allelic beacon placement at both B2M and CIITA loci.
  • AmpSeq was used to confirm the ddPCR data.
  • FIG.13 shows AmpSeq data for 6 iPSC lines including lines 1, 8, 9, 16, 17, and 22. The AmpSeq data did indeed confirm the ddPCR data from FIGs.
  • iPSC lines 16, 17 and 22 showed concurrent, bi-allelic beacon placement at both B2M and CIITA loci. Therefore, these lines were selected for programmable gene insertion (i.e., integration of donor polynucleotide templates) into one or both of the B2M locus and the CIITA locus. 8.4.
  • Example 3 Concurrent Integration of 6 kilobase donor templates at beacons incorporated into the B2M and CIITA loci using electroporation [0615] The iPS cell lines generated in Example 2 were used to assess concurrent integration integrate donor polynucleotide templates at the B2M locus, the CIITA locus, or both.
  • a first donor polynucleotide template (pDY-GG-EF1aGFP (SEQ ID NO: 618) about 6 kilobase (kb) in size having an AttP integration recognition site comprising a GG central dinucleotide was targeted to the AttB integration recognition site that was site-specifically integrated at the B2M locus.
  • This Example also included a second donor polynucleotide template (pL1113 (SEQ ID NO: 619) about 5 kb in size having an AttP integration recognition site comprising a GT central dinucleotide that was targeted to the AttB integration recognition site site- specifically integrated at the CIITA locus.
  • the undifferentiated iPS cell line (line #17) was electroporated with plasmid DNA encoding BxB1 as well as plasmid DNA from the first donor polynucleotide template and/or plasmid DNA from the second donor polynucleotide template.
  • plasmid DNA encoding BxB1 as well as plasmid DNA from the first donor polynucleotide template and/or plasmid DNA from the second donor polynucleotide template.
  • a single cell suspension of 100,000 iPSCs in 10 ⁇ L E1 buffer was combined with plasmid DNA according to the conditions specified in the Table 14.
  • Electroporation was performed using a Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 ⁇ M ROCK inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, the media was refreshed without Y27632 and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and beacon placement at the B2M locus and/or the CIITA locus was measured using ddPCR and/or AmpSeq.
  • FIGs.14A-B illustrates the primer and probe design for detecting integration of donor polynucleotide templates at the B2M locus (FIG. 14A) and the CIITA locus (FIG. 14B) using ddPCR.
  • FIG.15 shows integration of the first donor polynucleotide template at the B2M locus (site 1) and the second donor polynucleotide template at the CIITA locus (site 2) as determined by ddPCR.
  • FIG. 15 shows successful integration of the first donor polynucleotide template at the B2M locus (site 1) in both the single and duplex conditions.
  • FIG.15 also illustrates successful integration of the second donor polynucleotide template at the CIITA locus (site 2) in both the single and duplex conditions.
  • first “single” condition integration of the first donor polynucleotide template at the B2M locus (site 1) occurred at about 1% of the B2M locus (see FIG. 15).
  • second donor polynucleotide template at the CIITA locus (site 2) occurred at about 9% of the CIITA loci (see FIG.15).
  • Example 4 Integration of 31 kilobase adenovirus donor template within beacons incorporated into the CIITA locus in an iPSC
  • the iPS cell lines generated in Example 2 were used to test integration of a 31 kilobase (kb) donor polynucleotide template at the CIITA locus.
  • the donor polynucleotide template was a 31kb adenoviral vector (SEQ ID NO: 667) containing an AttP integration recognition site comprising a GG central dinucleotide that was integrated to the AttB integration recognition site site-specifically integrated at exon 2 of the CIITA locus.
  • the adenoviral donor template was introduced into the iPSCs as an adenovirus.
  • Adenovirus production was performed as described in Sicilnidi et al. (bioRxiv 2021.11.01.466786; doi.org/10.1101/2021.11.01.466786) and Wold et al. (Curr. Gene Ther.13, 421–433 (2013)), each of which are herein incorporated by reference in its entireties.
  • the undifferentiated iPS cell line was electroporated with plasmid DNA encoding BxB1.
  • a single cell suspension of 100,000 iPSCs in 10 ⁇ L E1 buffer was combined with 0.5 ⁇ g of plasmid DNA encoding BxB1.
  • Electroporation was performed using a Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse.
  • the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 0 ⁇ M ROCK inhibitor Y27632 and allowed to recover for about 24 hours.
  • iPSCs were contacted with adenovirus containing the donor polynucleotide template.
  • MOIs multiplicity of infections
  • iPSCs were cultured for an additional two days prior to harvesting the cells and analyzing for integration of the donor polynucleotide templates. DNA was harvested using a LGC QuickExtract buffer and integration of donor polynucleotide template at the CIITA locus was measured using ddPCR.
  • FIG.14B shows a schematic of the primer and probe design for the ddPCR assay used to detect integration of the 31kb donor polynucleotide template at the CIITA locus.
  • the 31kb donor polynucleotide template integrated into the CIITA locus in a dose-dependent manner.
  • the 31kb donor polynucleotide integrated into the CIITA locus occurred in about 6% of the CIITA locus.
  • AtgRNA used in this Example are shown in Table 15.
  • three forward atgRNAs and four reverse atgRNA all within the 200bp of CD52 exon 1 were designed. After identifying these atgRNAs, forward and reverse atgRNA were combined to make 12 combinations of dual atgRNAs pairs (see Table 15 and FIG.19A).
  • Each of these twelve atgRNA pairs were individually electroporated along with a mRNA encoding a gene editor polypeptide (e.g., a Cas9n-RT) into iPSC clone #17 (see FIG.19A for atgRNA pairs).
  • a gene editor polypeptide e.g., a Cas9n-RT
  • Electroporation 3.0 ⁇ g of an mRNA encoding the gene editor polypeptide and 0.5 ⁇ L (50pmol) of each of the atgRNA RNAF and atgRNA R were added to 100,000 cells of iPS clone #17. Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Clone #17 has perfect beacon placement in both B2M and CIITA loci. After electroporation, genomic DNA was isolated and ddPCR was performed using primers and proved designed to detect beacon placement within the 200bp of CD52 exon 1 (see Table 13). As shown in FIG.
  • ddPCR analysis revealed the highest performing atgRNA pair, which was pair 6, resulted in 39% beacon placement.
  • Other top performing atgRNA pairs included pairs 2, 5, and 8, each having beacon placement higher than 30% (see FIG.19A).
  • the highest performing pairs i.e., pairs 2, 5, 6 and 8 ( ⁇ 30% BP)
  • Cells electroporated with pairs 2, 6, and 8 were subjected to next generation sequencing as an alternative means for detecting beacon placement.
  • NGS analysis confirmed that pairs 2, 6, and 8 produced the highest beacon placement, and in fact had higher beacon placement percentages than were detected from ddPCR analysis.
  • atgRNA were designed to target additional regions of CD52.
  • CD52 is a small protein with only two exons, so additional guide atgRNAs are being designed and tested that target exon1-intron 1 junction, intron1-exon2 junction and exon 2. Similar to the atgRNA targeting CD52 exon 1, the atgRNA are designed such that they knockout expression of CD52 while also placing a beacon to be used for programmable gene insertion. In particular, the atgRNA designed to target the intron1-exon2 junction were designed to delete the coding sequence upstream of serine 36. CD52 has 61 amino acids where amino acids 1-24 are the signal peptide and amino acids 37-61 of the propeptide are cleaved . Therefore, as long as placing the beacon removes the first 35 amino acid residues of CD52 it will lose function.
  • iPSC line #17 was electroporated with an mRNA encoding the gene editor polypeptide and a pair of atgRNA that enable beacon placement (and subsequent knock out of) in the CD52 locus. Electroporated iPSCs were plated on Cell Basement Membrane-coated plates in Pluripotent Stem Cell SFM XF/FF media and allowed to recover.
  • iPSCs were plated at a density suitable to allow the single cells to grow into iPSC colonies that could be picked and expanded for purposes of cell line generation.
  • iPSCs were grown in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 ⁇ M ROCK inhibitor Y27632.
  • colonies were picked, disassociated into clumps (e.g., no less than 4 cells per clump) and re-plated onto a Cell Basement Membrane-coated dish.
  • the picked iPSC lines were expanded, analyzed for beacon placement, and cryopreserved. 8.7.
  • Example 6 Beacon placement at CISH locus in iPSCs having beacons in B2M and CIITA loci
  • a dual atgRNA approach was used for beacon (also referred to as an integration recognition site) placement at the CISH locus.
  • a non-limiting example of a method of generating an iPSC where the method includes placing a beacon in the CISH locus in an iPSC line already having beacons in each of the B2M and CIITA loci is shown in FIG. 8.
  • AtgRNA used in this Example are shown in Table 16. To knockout CISH and place a beacon in the CISH locus, six pairs of atgRNAs were designed that targeted exon 1 and exon 2 in the CISH locus. After identifying these atgRNAs, forward and reverse atgRNA were combined to make the six atgRNA pairs (see Table 16 and FIGs.20A-20B).
  • the six pairs of atgRNA were electroporated along with a mRNA encoding a gene editor polypeptide (e.g., a Cas9n-RT) into iPSC clone #17.
  • a gene editor polypeptide e.g., a Cas9n-RT
  • 3.0 ⁇ g of an mRNA encoding the gene editor polypeptide and 0.5 ⁇ L (50pmol) of each of the atgRNA RNAF and atgRNA R were added to 100,000 cells of iPSC clone #17. Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. As described herein, clone #17 has perfect beacon placement at both the B2M and the CIITA loci.
  • FIG. 20A ddPCR analysis revealed the highest performing atgRNA pair produced about 61% percent beacon placement.
  • gDNA from cells electroporated with the two highest performing atgRNA pairs were kept for NGS analysis to further characterize perfect beacon placement.
  • Cells electroporated in FIG.20A were also subjected to next generation sequencing as an alternative means for detecting beacon placement.
  • FIG. 20B which shows the NGS analysis, confirmed the same pattern of beacon placement efficiency shown in FIG.20A.
  • iPSC line #17 was electroporated with an mRNA encoding the gene editor polypeptide and a pair of atgRNA that enable beacon placement (and subsequent knock out of) in the CISH locus.
  • Electroporated iPSCs were plated on Cell Basement Membrane- coated plates in Pluripotent Stem Cell SFM XF/FF media and allowed to recover. iPSCs were plated at a density suitable to allow the single cells to grow into iPSC colonies that could be picked and expanded for purposes of cell line generation. During initial colony expansion (i.e., prior to picking), iPSCs were grown in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 ⁇ M ROCK inhibitor Y27632. About 7-14 days following plating, colonies were picked, disassociated into clumps (e.g., no less than 4 cells per clump) and re-plated onto a Cell Basement Membrane-coated dish.
  • clumps e.g., no less than 4 cells per clump
  • Example 7 Beacon placemen at ADORA2A locus in iPSC having beacons in B2M and CIITA loci
  • a dual atgRNA approach was used for beacon placement at the ADORA2A locus.
  • FIG.8 A non-limiting example of a method of generating an iPSC where the method includes placing a beacon in the ADORA2A locus in an iPSC line already having beacons in the B2M and CIITA loci is shown in FIG.8.
  • AtgRNA used in this Example are shown in Table 17.
  • AtgRNA pairs were designed that targeted exon 2 of the ADOR2A2 locus. Forward and reverse atgRNA were combined to make the five atgRNA pairs that were tested in this example (see Table 17 and FIGs.21A-21B).
  • the five pairs of atgRNA were electroporated along with a mRNA encoding a gene editor polypeptide (e.g., a Cas9n-RT) into iPSC clone #17.
  • a gene editor polypeptide e.g., a Cas9n-RT
  • 3.0 ⁇ g of an mRNA encoding the gene editor polypeptide and 0.5 ⁇ L (50pmol) of each of the atgRNA RNAF and atgRNA R were added to 100,000 cells of iPS clone #17. Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse.
  • FIG. 21A ddPCR analysis revealed the highest performing atgRNA produced about 58% percent beacon placement.
  • FIG. 21B which shows the NGS analysis, confirmed the same pattern of beacon placement efficiency shown in FIG.21A.
  • iPSC line #17 was electroporated with an mRNA encoding the gene editor polypeptide and a pair of atgRNA that enable beacon placement (and subsequent knock out of) in the ADORA2A locus.
  • Electroporated iPSCs were plated on Cell Basement Membrane-coated plates in Pluripotent Stem Cell SFM XF/FF media and allowed to recover. iPSCs were plated at a density suitable to allow the single cells to grow into iPSC colonies that could be picked and expanded for purposes of cell line generation. During initial colony expansion (i.e., prior to picking), iPSCs were grown in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 ⁇ M ROCK inhibitor Y27632. About 7-14 days following plating, colonies were picked, disassociated into clumps (e.g., no less than 4 cells per clump) and re-plated onto a Cell Basement Membrane-coated dish.
  • clumps e.g., no less than 4 cells per clump
  • Example 8 Concurrent beacon placement at B2M, CIITA, and CD52 loci in iPSCs using a dual atgRNA approach at each locus
  • a dual atgRNA approach to beacon placement was used for beacon placement at each locus.
  • the same dual atgRNAs as described in Example 1 (B2M and CIITA atgRNAs) and Example 5 (CD52 atgRNAs) were used in this Example.
  • FIG.8 A non-limiting example of a method of generating an iPSC where the method includes concurrent site-specific integration of integration recognition sites in the B2M locus, the CIITA locus, and the CD52 locus is shown in FIG.8.
  • iPSC lines ACS-1030 (iPSC1) and ACS-1026 (iPSC2) were cultured as undifferentiated iPSCs on Cell Basement Membrane (ATCC)-coated plates in Pluripotent Stem Cell SFM XF/FF media (ATCC). Undifferentiated iPSCs were electroporated with 2 ⁇ g mRNA encoding a gene editor polypeptide (Cas9n-RT) and 50 picomoles of each atgRNA as described in Table 18.
  • PE2 mRNA plus B2M bmp003, CIITA bmp003 and CD52 mod2 atgRNA pairs were electroporated into WT iPSC2 cells.
  • Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 ⁇ M ROCK-inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, media was changed and cells were cultured for an additional 72 hours.
  • FIG. 22 shows ddPCR data of percent beacon placement.
  • the ddPCR data revealed that the beacon placement efficiency is comparable between triple and single beacon placement (see FIG.22). For B2M the efficiency was about 80%, for CIITA the efficiency was between 10- 20%, and for CD52 the efficiency was about 50%.
  • B2M the efficiency was about 80%
  • CIITA the efficiency was between 10- 20%
  • CD52 the efficiency was about 50%.
  • this data showed concurrent beacon placement in three different target loci (i.e., B2M, CIITA, and CD52 loci) following a single transfection in wild type iPSC. 8.10.
  • Example 9 Concurrent beacon placement at the TRAC and the AAVS1 loci using a dual atgRNA approach at each locus
  • attB beacon placement i.e., integration recognition site placement or incorporation
  • iPSC clone 17 i.e., an iPSC clone having beacons already placed at the B2M and CIITA loci
  • a dual atgRNA approach to beacon placement was used for beacon placement at each locus.
  • AtgRNAs for targeting the TRAC and AAVS1 loci are as described in Table 19.
  • FIG.8 A non-limiting example of a method of generating an iPSC where the method includes concurrent site-specific integration of integration recognition sites in the B2M locus, the CIITA locus, the TRAC locus, and the AAVS1 locus is shown in FIG.8.
  • iPSC lines 16, 17, and 29 were cultured as undifferentiated iPSCs on Cell Basement Membrane (ATCC)-coated plates in Pluripotent Stem Cell SFM XF/FF media (ATCC). Undifferentiated iPSCs were electroporated with 2 ⁇ g mRNA encoding a gene editor polypeptide (Cas9n-RT) and 50 picomoles of each atgRNA.
  • Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 ⁇ M ROCK-inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, media was changed and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and beacon placement at the B2M locus and/or the CIITA locus was measured using ddPCR and/or AmpSeq. [0648] FIG.
  • FIG. 23 shows concurrent beacon placement at the TRAC and AAVS1 loci in iPSC clone 17.
  • FIG. 23 shows ddPCR data of percent beacon placement with the respective pairs with nearly 100% beacon placement at AAVS1, and around 30% beacon placement at TRAC.
  • this data demonstrated successful generation of a cell line comprising four beacons placed at four different loci in the genome. 8.11.
  • Example 10 iPSC line generation of iPSC containing beacons in B2M, CIITA, TRAC and AAVS1 loci
  • iPSC line #17 which has a beacon already placed in both the B2M and CIITA loci, was electroporated with dual atgRNAs targeting the TRAC and AAVS1 are described in the preceding examples.
  • FIG.24 A non-limiting example of a method of generating an iPSC cell line where the method includes concurrent site-specific integration of integration recognition sites (beacons) in one or more of the B2M locus, the CIITA locus, the TRAC locus, and the AAVS1 locus is shown in FIG.24.
  • integration recognition sites beacons
  • FIG.24 A non-limiting example of a method of generating an iPSC cell line where the method includes concurrent site-specific integration of integration recognition sites (beacons) in one or more of the B2M locus, the CIITA locus, the TRAC locus, and the AAVS1 locus is shown in FIG.24.
  • iPSC clone # 17 was cultured as undifferentiated iPSCs on Cell Basement Membrane (ATCC)-coated plates in Pluripotent Stem Cell SFM XF/FF media (ATCC).
  • Undifferentiated iPSCs were electroporated with 2 ⁇ g mRNA encoding a gene editor polypeptide (Cas9n-RT) and atgRNA as described in Examples 2 and 9.
  • electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse.
  • the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 ⁇ M ROCK-inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, media was changed and cells were cultured for an additional 72 hours.
  • FIG. 25 shows next generation sequencing data for beacon placement at the TRAC and AAVS1 loci in the indicated iPSC lines. Overall, this data demonstrated successful generation of a cells comprising four beacons placed at four different loci in the genome.
  • iPSC lines containing beacons in each the B2M, CIITA, TRAC, and AAVS1 loci were used for multiplexed programmable gene insertion (i.e., concurrent site-specific integration of four donor polynucleotide templates).
  • Example 11 Concurrent site-specific integration of four donor templates in iPS cells containing beacons at B2M, CIITA, TRAC, and AAVS1 loci
  • iPS cells generated in Example 10 containing beacons at each of the B2M, CIITA, TRAC, and AAVS1 loci were used to assess concurrent integration of donor polynucleotide templates at each of the locus (also referred to as multiplexed programmable gene insertion).
  • Each of the four donor polynucleotide templates included a beacon (i.e., an integration recognition site (e.g., an AttP site)) comprising a different central dinucleotide than the beacons in the other donor polynucleotide templates.
  • Each beacon in each donor polynucleotide template was “paired” with its corresponding (cognate) integration recognition site site-specifically integrated into the iPSC genome at one of the B2M, CIITA, TRAC, and AAVS1 loci (see lines generated in Example 10), such that when contacted with an integration enzyme a recombination event occurs and integration results.
  • a first donor polynucleotide template (pDY-GG-EF1aGFP (SEQ ID NO: 618)) about 6 kilobase (kb) in size and having an AttP integration recognition site comprising a GG central dinucleotide was targeted to the AttB integration recognition site that was site-specifically integrated at the B2M locus.
  • the second donor polynucleotide template (pL1113 (SEQ ID NO: 619)) was about 5 kb in size and included an AttP integration recognition site comprising a GT central dinucleotide that was targeted to the AttB integration recognition site site- specifically integrated at the CIITA locus.
  • the third donor polynucleotide template (pL1220 (SEQ ID NO: 620)) was about 3.8 kb in size and included an AttP integration recognition site comprising a CT central dinucleotide that was targeted to the AttB integration recognition site site-specifically integrated at the TRAC locus.
  • the fourth donor polynucleotide template (pL1218 (SEQ ID NO: 621)) was about 4.0 kb in size and included an AttP integration recognition site comprising an AC central dinucleotide that was targeted to the attB integration recognition site site-specifically integrated at the AAVS1 locus. Sequences of each the donor polynucleotide templates used for multiplexed programmable gene insertion are described in Table 20.
  • each donor polynucleotide template (about 3.2 ⁇ g total) were combined with about 1.0 ⁇ g of a plasmid PL193 encoding a BxB1 polypeptide.
  • the final volume was brought to 6 ⁇ L and combined with about 100,000 cells of iPSC clones #16 or clone #29 in 10 ⁇ L E1 buffer. Similar to clone #17, clones #16 and #29 each have beacons integrated at the B2M, CIITA, TRAC, and AAVS1 loci.
  • the cells-plasmid mixture was electroporated using a Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 ⁇ M ROCK inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, the media was refreshed without Y27632 and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and integration of the donor polynucleotides at each locus was measured using ddPCR and/or AmpSeq.
  • the final volume was brought to 6 ⁇ L and combined with about 100,000 cells of iPSC clones #30 in 10 ⁇ L E1 buffer. Similar to clone #17, clone #30 has beacons integrated at the B2M, CIITA, TRAC, and AAVS1 loci.
  • the cells-plasmid mixture was electroporated using a Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 ⁇ M ROCK inhibitor Y27632 and allowed to recover for about 24 hours.

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Abstract

Described herein is a method of editing a pleiopluripotent stem cell genome. In typical embodiments, an integration target recognition site (i.e., attB or attP site) is incorporated (i.e., beacon placement) into a pleiopluripotent stem cell genome by delivering into the cell a gene editor (or a prime editor fusion), attachment site-containing guide RNA (atgRNA), and, optionally, a nicking guide RNA (ngRNA). Further, integrating a donor polynucleotide template into the pluripotent stem cell genome at the incorporated target recognition site is described herein thereby producing a genetically modified pluripotent stem cell.

Description

PLEIOPLURIPOTENT STEM CELL PROGRAMMABLE GENE INSERTION 1. CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Application No.63/391,659, filed July 22, 2022 and U.S. Provisional Application No.63/375,190, filed September 9, 2022; each of which is hereby incorporated in its entirety by reference. 2. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing with 667 sequences, which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on July 21, 2023 is named 52810PCT-sequencelisting.xml, and is 918,523 bytes in size. 3. BACKGROUND [0003] Programmable, efficient, and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. [0004] There is a need in the art for techniques and gene editor compositions and gene editor guide RNAs which address and overcome these shortcomings and enable the programmable genomic integration of a large gene(s) in pluripotent stem cells. 4. SUMMARY [0005] This disclosure features a method of generating a pleiopluripotent cell, pleiopluripotent cells, pleiopluripotent-derived cells, and methods of using the same. In one aspect, this disclosure features a method of generating a pleiopluripotent cell, where the method includes site-specifically incorporating at least a first integration recognition site (e.g., AttB) into the genome of a pleiopluripotent cell, where site-specifically incorporating the genome of a pluripotent cell is effected by introducing into the pleiopluripotent stem cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein or a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the pleiopluripotent stem cell genome at sites respectively flanking the specific incorporation site, and at least one of the first paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site-specifically incorporated into the genome of the pleiopluripotent stem cell. In some embodiments, the first of the two paired guide RNAs is a first atgRNA that further includes a first RT template that comprises at least a portion of the first integration recognition site; and a second of the two paired guide RNAs is a second atgRNA that further includes a second RT template that comprises at least a portion of the first integration recognition site, wherein the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site. [0006] In another aspect, the method includes integrating at least a first donor polynucleotide template into the pleiopluripotent cell genome at the first incorporated integration recognition site, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the at least one incorporated genomic integration recognition site by the integrase; thereby producing a second generation pleiopluripotent cell. [0007] In another aspect, this disclosure features a pleiopluripotent cell comprising at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome. In another aspect, this disclosure features a pleiopluripotent cell comprising a donor polynucleotide template comprised of one or more orthogonal integration recognition sites integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0008] In another aspect, this disclosure features a pleiopluripotent cell or a population thereof, where (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site incorporated site-specifically into the PC- derived cell genome. In another aspect, this disclosure features. [0009] In another aspect, this disclosure features a pleiopluripotent cell or a population thereof, wherein (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC- derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0010] The present disclosure provides nucleic acid compositions, methods, and an overall platform for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE) (see Ionnidi et al.; Nat. Biotech.41: 500–512 (2023)); the entirety of which is incorporated herein by reference), transposon-mediated gene editing, or other suitable gene editing, or gene incorporation technology. Non-limiting examples of PASTE are also described in U.S. Pat. 11,572,556 and PCT Publication Nos. WO 2023/077148A1 and WO 2023/122764, each of which are hereby incorporated by reference in their entireties. [0011] In one aspect, this disclosure features a method of generating a pleiopluripotent cell, the method comprising: (a) site-specifically incorporating at least a first integration recognition site into a genome of a pleiopluripotent cell. In some embodiments of the methods, site-specifically incorporating the at least first integration recognition site is effected by introducing into the pleiopluripotent cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein or a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the pleiopluripotent cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site-specifically incorporated into the genome of the pleiopluripotent cell. [0012] In some embodiments, the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is a first atgRNA that further includes a first RT template that comprises at least a portion of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a second atgRNA that further includes a second RT template that comprises at least a portion of the first integration recognition site, wherein the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site. [0013] In some embodiments of the methods, the at least first pair of guide RNAs comprise: [0014] (i) the first of the two paired guide RNAs is an atgRNA that further includes an RT template that comprises at least a portion of the first integration recognition site, wherein the atgRNA encodes the entirety of the first integration recognition site; and [0015] (ii) a second of the two paired guide RNAs is a nicking gRNA. [0016] In some embodiments, the method further comprises incorporating a plurality of integration recognition sites. [0017] In some embodiments, the method further comprises: (b) integrating at least a first donor polynucleotide template into the pleiopluripotent cell genome at the first incorporated integration recognition site, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a second generation pleiopluripotent cell. [0018] In some embodiments of the method, steps (a) and (b) are performed concurrently. [0019] In some embodiments of the method, step (a) is performed prior to step (b). [0020] In another aspect, this disclosure features a method of generating a pleiopluripotent cell, the method comprising: (a) integrating, into the genome of any of the pleiopluripotent cells described herein at the first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a second generation pleiopluripotent cell. [0021] In some embodiments, the method further comprises (b) site-specifically incorporating a second integration recognition site into the genome of the pleiopluripotent cell, thereby generating a third generation pleiopluripotent cell. [0022] In some embodiments, the method further comprises: (c) integrating a second donor polynucleotide template into the pleiopluripotent cell genome at the second incorporated integration recognition site, by delivering into the cell: (i) the second donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration recognition sites orthogonal to the second integration recognition site, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the second incorporated genomic integration recognition sites by the integrase; thereby producing a fourth generation pleiopluripotent cell line. [0023] In some embodiments of the method, steps (a), (b), and (c) are performed concurrently. [0024] In some embodiments of the method, step (a) is performed prior to steps (b) and (c), wherein steps (b) and (c) are performed concurrently or step (b) is performed prior to step (c). [0025] In some embodiments of the method, the step of site-specifically incorporating the first integration recognition site and the step of site-specifically incorporating the second integration recognition site are performed concurrently. [0026] In some embodiments of the method, the first RT template encodes a first single-stranded DNA sequence and the second RT template encodes a second single-stranded DNA sequence. [0027] In some embodiments of the method, the first single-stranded DNA sequence comprises a complementary region with the first single-stranded DNA sequence. [0028] In some embodiments of the method, the first single-stranded DNA sequence and the first single-stranded DNA sequence form a duplex. [0029] In some embodiments of the method, the complementary region is 5 or more consecutive bases. [0030] In some embodiments of the method, the complementary region is 10 or more consecutive cases. [0031] In some embodiments of the method, the complementary region is 20 or more consecutive bases. [0032] In some embodiments of the method, the complementary region is 30 or more consecutive bases. [0033] In some embodiments of the method, at least one of the two paired guide RNAs has a chemical modification. [0034] In some embodiments of the method, the paired guide RNAs each have a chemical modification. [0035] In some embodiments of the method, at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a safe harbor locus. [0036] In some embodiments of the method, at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell. [0037] In some embodiments of the method, at least one of the at least first integration recognition sites is incorporated into the genome at a plurality of loci, wherein disruption of at least one of the loci is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell. [0038] In some embodiments of the method, at least one of the at least first integration recognition sites is incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof. [0039] In some embodiments of the method, the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. In some embodiments of the method, the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ. In some embodiments of the method, the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus. In some embodiments of the method, the locus is the B2M locus. In some embodiments of the method, the locus is the CIITA locus. [0040] In some embodiments of the method, the introducing step is performed by electroporation. In some embodiments of the method, introducing comprises electroporating a gene editor protein or a polynucleotide encoding a gene editor protein, a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein, at least a first pair or guide RNAs, the donor polynucleotide template, or a combination thereof into the pleiopluripotent cell occurs using one or more of a recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, anellovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, mRNA, RNP, or lipid nanoparticle. [0041] In some embodiments of the method, the introducing step is performed by transfection. In some embodiments of the method, introducing comprises transfecting mRNA encoding the gene editor protein, the prime editor fusion protein, the at least first pair of guide gRNAs, the donor polynucleotide template, or a combination thereof into the pleiopluripotent cell, wherein the donor polynucleotide template is selected from a mini circle, a nanoplasmid, and a miniDNA. [0042] In some embodiments of the method, at least one of the at least first integration recognition sites is specific for a serine integrase. [0043] In some embodiments of the method, at least one of the at least first integration recognition sites is an attB or attP site. [0044] In some embodiments of the method, at least one of the at least first integration recognition sites is a modified attB or attP site. [0045] In some embodiments of the method, at least one of the at least first integration recognition sites is specific for BxB1 or a modified BxB1. [0046] In some embodiments of the method, at least one of the at least first integration recognition sites is comprised of 38 or 46 nucleotides. [0047] In some embodiments of the method, the first donor polynucleotide template encodes one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof. [0048] In some embodiments of the method, the inducing the at least one of the one or more inducible suicide genes targets the pleiopluripotent cell or a cell matured from the pleiopluripotent cell for cell death. [0049] In some embodiments of the method, expression of at least of the one or more inducible suicide gene is driven by a pluripotent stem cell-specific promoter. In some embodiments of the method, expression of at least one of the one or more inducible suicide gene is driven by a constitutive promoter. In some embodiments of the method, expression of at least one of the one or more inducible suicide gene is driven by an inducible promoter. [0050] In some embodiments of the method, the one or more inducible suicide genes is selected from: caspase9, cytosine deaminase, and thymidine kinase. In some embodiments of the method, the one or more inducible suicide genes is a controllable caspase9. In some embodiments of the method, AP20187 (or an analog thereof) controls activity of Caspase9 or AP21967 (or an analog thereof) controls activity of Caspase9. In some embodiments, the method further comprises a second inducible suicide gene, wherein the second inducible suicide gene comprises a thymidine kinase. In some embodiments of the method, the polynucleotide encoding for the thymidine kinase is operably linked to the controllable capase9 protein using a 2A self-cleaving peptide or an internal ribosome entry site. [0051] In some embodiments of the method, the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells. [0052] In some embodiments of the method, the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells by inhibiting, blocking, or preventing recognition of the pleiopluripotent cell by macrophages, T- cells, Natural Killer (NK)-cells, antigen-presenting cells, or a combination thereof. [0053] In some embodiments of the method, the one or more exogenous polypeptides capable of modulating an immune response are capable of serving as a safety switch target. [0054] In some embodiments of the method, expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a pluripotent stem cell-specific promoter. [0055] In some embodiments of the method, expression of least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a constitutive promoter. [0056] In some embodiments of the method, expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by an inducible promoter. [0057] In some embodiments of the method, the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL-1, B2M-HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR. [0058] In some embodiments of the method, the donor polynucleotide template encodes a CD47 polypeptide, a PDL1 polypeptide, and a B2M-HLA-E polypeptide. [0059] In some embodiments of the method, each of the sequences coding for CD47, PDL1 and B2M-HLA-E are separated by a sequence coding for a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof. [0060] In some embodiments of the method, the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag. [0061] In some embodiments of the method, the donor polynucleotide template further comprises an additional orthogonal integrase target recognition site. [0062] In some embodiments of the method, the second integration recognition site is site- specifically incorporated into a safe harbor locus. [0063] In some embodiments of the method, the second integration recognition site is different from the at least first integration recognition attB or attP site. [0064] In some embodiments of the method, the second integration recognition site is specific for BxB1 or a modified BxB1. [0065] In some embodiments of the method, the second integration recognition sites is comprised of 38 or 46 nucleotides. [0066] In some embodiments of the method, a second donor polynucleotide template is integrated into the pleiopluripotent cell genome at the second integration recognition site site-specifically incorporated into the pleiopluripotent cell genome. [0067] In some embodiments of the method, the second donor polynucleotide template encodes one or more therapeutic agents. [0068] In some embodiments of the method, the one or more therapeutic agents is selected from: transcription factors, receptors, signaling molecules, secreted proteins, cytokines, enzymes, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, and persistence and/or survival of the iPSCs or cells derived from the iPSC. [0069] In some embodiments of the method, one or more therapeutic agents is a HLA class I protein. [0070] In some embodiments of the method, the one or more therapeutic agents is a HLA class II protein. [0071] In some embodiments of the method, the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents. [0072] In some embodiments of the method, the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agents. [0073] In some embodiments of the method, the second donor polynucleotide template comprises an inducible promoter operably linked to at least one of the one or more therapeutic agents. [0074] In some embodiments of the method, the first donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 1 kilobase (kb), at least 2 kb, at least 3kb, at least4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, at least 30kb, at least 31kb, at least 32kb, at least 33kb, at least 34kb, or at least 35kb or more. [0075] In some embodiments of the method, the first donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 30kb. [0076] In some embodiments of the method, the second donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 1 kilobase (kb), at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, at least 30kb, at least 31kb, at least 32kb, at least 33kb, at least 34kb, or at least 35kb or more. [0077] In some embodiments of the method, the second donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 30kb. [0078] In some embodiments of the method, the pleiopluripotent cell is a pluripotent stem cell. [0079] In some embodiments of the method, the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. [0080] In some embodiments of the method, the pluripotent stem cell is an induced pluripotent stem cell. [0081] In some embodiments of the method, the induced pluripotent stem cell is a human induced pluripotent stem cell. [0082] In some embodiments, the method further comprises site-specifically excising from the genome of the pleiopluripotent cell the first donor polynucleotide template, the second donor polynucleotide template, or both. [0083] In some embodiments of the method, wherein the site-specifically excising is effected by introducing into the pleiopluripotent cell an integrase that recognizes the one or more orthogonal integration recognition sites in the first donor polynucleotide template, the second donor polynucleotide template, or both. [0084] In some embodiments, the method further comprises cryopreserving the pleiopluripotent cell or a population thereof. [0085] In another aspect, this disclosure features a pleiopluripotent cell or population thereof generated using the method any of the methods described herein. [0086] In another aspect, this disclosure features a second generation pleiopluripotent cell generated using the method of any of methods described herein. [0087] In another aspect, this disclosure features a third generation pleiopluripotent cell generated using the method of any of the methods described herein. [0088] In another aspect, this disclosure features a fourth generation pleiopluripotent cell generated using the method of any of the methods described herein. [0089] In another aspect, this disclosure features a pleiopluripotent cell, comprising: [0090] at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome. [0091] In another aspect, this disclosure features a pleiopluripotent cell comprising: [0092] a donor polynucleotide template comprised of one or more orthogonal integration recognition site integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0093] In some embodiments of the pleiopluripotent cell, wherein at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a safe harbor locus. [0094] In some embodiments of the pleiopluripotent cell, at least one of the at least first integration recognition sites is site-specifically incorporated within a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell. [0095] In some embodiments of the pleiopluripotent cell, at least one of the at least first integration recognition sites is site-specifically incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof. In some embodiments, the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. [0096] In some embodiments of the pleiopluripotent cell, the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ. [0097] In some embodiments of the pleiopluripotent cell, the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA- DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus. [0098] In some embodiments, the locus is the B2M locus. In some embodiments, the locus is the CIITA locus. [0099] In some embodiments of the pleiopluripotent cell, at least one of the at least first integration recognition sites is specific for a serine integrase. [0100] In some embodiments of the pleiopluripotent cell, at least one of the at least first integration recognition sites is an attB or attP site. [0101] In some embodiments of the pleiopluripotent cell, at least one of the at least first integration recognition sites is a modified attB or attP site. [0102] In some embodiments of the pleiopluripotent cell, at least one of the at least first integration recognition sites is specific for BxB1 or a modified BxB1. [0103] In some embodiments of the pleiopluripotent cell, at least one of the at least first integration recognition site is comprised of 38 or 46 nucleotides. [0104] In some embodiments of the pleiopluripotent cell, the donor polynucleotide template encodes one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof. In some embodiments, inducing at least one of the one or more inducible suicide genes targets the pleiopluripotent cell or a cell matured from the pleiopluripotent cell for cell death. In some embodiments, expression of at least one of the one or more inducible suicide gene is driven by a pluripotent stem cell-specific promoter. In some embodiments, expression of at least one of the one or more inducible suicide gene is driven by a constitutive promoter. In some embodiments, expression of at least one of the one or more inducible suicide gene is driven by an inducible promoter. In some embodiments, the one or more inducible suicide genes is selected from: caspase9, cytosine deaminase, and thymidine kinase. In some embodiments, the one or more inducible suicide genes is a controllable caspase9. [0105] In some embodiments, AP20187 (or analog thereof) controls activity of Caspase9 or AP21967 (or analog thereof) controls activity of Caspase9. [0106] In some embodiments, the pleiopluripotent cell further comprises a second inducible suicide gene. In some embodiments, the second inducible suicide gene comprises a thymidine kinase. In some embodiments, the polynucleotide encoding for the thymidine kinase is operably linked to the controllable capase9 protein using a 2A self-cleaving peptide or an internal ribosome entry site. [0107] In some embodiments of the pleiopluripotent cell, the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells. In some embodiments, the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells by inhibiting, blocking, or preventing recognition of the PC-derived cell by macrophages, T-cells, Natural Killer (NK)-cells, antigen-presenting cells, or a combination thereof. [0108] In some embodiments of the pleiopluripotent cell, the one or more exogenous polypeptides capable of modulating an immune response are capable of serving as a safety switch target. [0109] In some embodiments of the pleiopluripotent cell, expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a pluripotent stem cell-specific promoter. [0110] In some embodiments of the pleiopluripotent cell, expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a constitutive promoter. [0111] In some embodiments of the pleiopluripotent cell, expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by an inducible promoter. [0112] In some embodiments of the pleiopluripotent cell, the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL-1, B2M-HLA-E, HLA- G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof. In some embodiments, the donor polynucleotide template encodes a CD47, PDL1, and B2M-HLA- E. In some embodiments, the polynucleotide template encoding CD47, PDL1 and B2M-HLA-E are separated by a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof. [0113] In some embodiments of the pleiopluripotent cell, the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag. In some embodiments, the tag is an ALFA tag. [0114] In some embodiments, the pleiopluripotent cell comprises two integration recognition sites, three integration recognition sites, four integration recognition sites, five integration recognition sites, six integration recognition sites, seven integration recognition sites, eight integration recognition sites, nine integration recognition sites, or ten or more integration recognition sites. [0115] In some embodiments, the pleiopluripotent cell further comprises a second integration recognition site site-specifically incorporated into the pleiopluripotent cell genome. In some embodiments, the second integration recognition sites is site-specifically incorporated into a safe harbor locus. In some embodiments, the second integration recognition site is site-specifically incorporated within a second locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell. In some embodiments, the second integration recognition site is site-specifically incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof. In some embodiments, the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. In some embodiments, the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ. In some embodiments, the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus. In some embodiments, the locus is the B2M locus. In some embodiments, the locus is the CIITA locus. [0116] In some embodiments of the pleiopluripotent cell, at least one of the at least second integration recognition sites is specific for a serine integrase. [0117] In some embodiments of the pleiopluripotent cell, the second integration recognition sites is different from the at least first integration recognition attB or attP site. [0118] In some embodiments of the pleiopluripotent cell, the second integration recognition sites is a modified attB or attP site. [0119] In some embodiments of the pleiopluripotent cell, the second integration recognition site is specific for BxB1 or a modified BxB1. [0120] In some embodiments of the pleiopluripotent cell, the second integration recognition sites is comprised of 38 or 46 nucleotides. [0121] In some embodiments of the pleiopluripotent cell, the second donor polynucleotide template encodes one or more therapeutic agents. In some embodiments, the one or more therapeutic agents is selected from: transcription factors, receptors, signaling molecules, secreted proteins, cytokines, enzymes, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, and persistence and/or survival of the iPSCs or cells derived from the iPSC. In some embodiments, the one or more therapeutic agents is a HLA class I proteins. In some embodiments, the one or more therapeutic agents is a HLA class II proteins. In some embodiments, wherein the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents. [0122] In some embodiments of the pleiopluripotent cell, the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agent. [0123] In some embodiments of the pleiopluripotent cell, the second donor polynucleotide template comprises an inducible promoter operably linked to at least one of the one or more therapeutic agent. [0124] In some embodiments of the pleiopluripotent cell, the pleiopluripotent cell is a pluripotent stem cell. [0125] In some embodiments of the pleiopluripotent cell, the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. [0126] In some embodiments of the pleiopluripotent cell, the pluripotent stem cell is an induced pluripotent stem cell. [0127] In some embodiments of the pleiopluripotent cell, the induced pluripotent stem cell is a human induced pluripotent stem cell. [0128] In another aspect, this disclosure features a composition comprising a clonal population of any of the pleiopluripotent cells described herein. [0129] In another aspect, this disclosure features a pharmaceutical composition comprising a clonal population of pleiopluripotent cells described herein and a pharmaceutically acceptable excipient or carrier. [0130] In another aspect, this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site is incorporated site-specifically into the PC-derived cell genome. [0131] In another aspect, this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0132] In another aspect, this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a hematopoietic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site is incorporated site-specifically into the PC-derived cell genome. [0133] In another aspect, this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a hematopoietic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0134] In some embodiments of the PC-derived cell or a population thereof, the hematopoietic cell is selected from: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells. [0135] In another aspect, this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a neuronal cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site integration recognition sites incorporated site-specifically into the PSC-derived cell genome. [0136] In another aspect, this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a neuronal cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0137] In some embodiments of the PC-derived cell or a population thereof, the neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron. [0138] In another aspect, this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a cardiac cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site incorporated site-specifically into the PSC-derived cell genome. [0139] In another aspect, this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a cardiac cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0140] In some embodiments of the PC-derived cell or a population thereof, the cardiac cell is selected from: a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte. [0141] In another aspect, this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a pancreatic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site incorporated site-specifically into the PC-derived cell genome. [0142] In another aspect, this disclosure features a pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a pancreatic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0143] In some embodiments of the PC-derived cell or a population thereof, the pancreatic cell is selected from a pancreatic progenitor cell, pancreatic endoderm, a endocrine pancreatic cell, an exocrine pancreatic cell, a islet progenitor, and a beta cell. [0144] In some embodiments of the PC-derived cell or a population thereof, the PC-derived cell further comprises two integration recognition sites, three integration recognition sites, four integration recognition sites, five integration recognition sites, six integration recognition sites, seven integration recognition sites, eight integration recognition sites, nine integration recognition sites, or ten or more integration recognition sites. [0145] In some embodiments of the PC-derived cell or a population thereof, at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a safe harbor locus. [0146] In some embodiments of the PC-derived cell or a population thereof, at least one of the at least first integration recognition sites is site-specifically incorporated within a locus, disruption of which is capable of reducing allogeneic immune response to the PC-derived cell. [0147] In some embodiments of the PC-derived cell or a population thereof, at least one of the at least first integration recognition sites is incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof. [0148] In some embodiments of the PC-derived cell or a population thereof, the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. [0149] In some embodiments of the PC-derived cell or a population thereof, the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ. [0150] In some embodiments of the PC-derived cell or a population thereof, the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA- DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus. In some embodiments, the locus is the B2M locus. In some embodiments, the locus is the CIITA locus. [0151] In some embodiments of the PC-derived cell or a population thereof, at least one of the at least first recognition sites is specific for a serine integrase. [0152] In some embodiments of the PC-derived cell or a population thereof, at least one of the at least first integration recognition sites is an attB or attP site. [0153] In some embodiments of the PC-derived cell or a population thereof, at least one of the at least first integration recognition sites is a modified attB or attP site. [0154] In some embodiments of the PC-derived cell or a population thereof, at least one of the at least first integration recognition site is specific for BxB1 or a modified BxB1. [0155] In some embodiments of the PC-derived cell or a population thereof, at least one of the at least first integration recognition sites is comprised of 38 or 46 nucleotides. [0156] In some embodiments of the PC-derived cell or a population thereof, the first donor polynucleotide template encodes one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof. In some embodiments, inducing at least one of the one or more inducible suicide genes targets the pleiopluripotent cell or a cell matured from the pleiopluripotent cell for cell death. In some embodiments, expression of at least one of the one or more inducible suicide gene is driven by a pluripotent stem cell-specific promoter. In some embodiments, expression of the at least one of the one or more inducible suicide genes is driven by a constitutive promoter. In some embodiments, expression of the at least one of the one or more inducible suicide genes is driven by an inducible promoter. In some embodiments, the one or more inducible suicide genes is select from: caspase9, cytosine deaminase, and thymidine kinase. In some embodiments, the one or more inducible suicide genes is a controllable caspase9. In some embodiments, AP20187 (or analog thereof) controls activity of Caspase9 or AP21967 (or analog thereof) controls activity of Caspase9. [0157] In some embodiments of the PC-derived cell or a population thereof, the cell further comprises a second inducible suicide gene. In some embodiments, the polynucleotide encoding for the second inducible suicide gene is operably linked to the controllable capase9 protein using a 2A self-cleaving peptide or an internal ribosome entry site. In some embodiments, the second inducible suicide gene comprises a thymidine kinase. [0158] In some embodiments of the PC-derived cell or a population thereof, the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells. In some embodiments, the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells by inhibiting, blocking, or preventing recognition of the PC-derived cell by macrophages, T- cells, Natural Killer (NK)-cells, antigen-presenting cells, or a combination thereof. In some embodiments, the one or more exogenous polypeptides capable of modulating an immune response are capable of serving as a safety switch target. [0159] In some embodiments of the PC-derived cell or a population thereof, expression of at least one of the one or more exogenous polypeptide capable of modulating an immune response is driven by a pluripotent stem cell-specific promoter. [0160] In some embodiments of the PC-derived cell or a population thereof, expression of at least one of the one or more exogenous polypeptide capable of modulating an immune response is driven by a constitutive promoter. [0161] In some embodiments of the PC-derived cell or a population thereof, expression of at least one of the one or more exogenous polypeptide capable of modulating an immune response is driven by an inducible promoter. [0162] In some embodiments of the PC-derived cell or a population thereof, the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL- 1, B2M-HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof. In some embodiments, the donor polynucleotide template encodes a CD47, PDL1, and B2M-HLA-E. In some embodiments, the polynucleotide template encoding CD47, PDL1 and B2M-HLA-E are separated by a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof. [0163] In some embodiments of the PC-derived cell or a population thereof, the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag. In some embodiments, the tag is an ALFA tag. [0164] In some embodiments of the PC-derived cell or a population thereof, the cell further comprises a second integration recognition site site-specifically incorporated into the pleiopluripotent cell genome. In some embodiments, the second integration recognition sites is site-specifically incorporated into a safe harbor locus. In some embodiments, the second integration recognition site is site-specifically incorporated within a second locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell. In some embodiments, the second integration recognition site is site-specifically incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof. In some embodiments, the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA- DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus. In some embodiments, the locus is the B2M locus. In some embodiments, the locus is the CIITA locus. [0165] In some embodiments of the PC-derived cell or a population thereof, the second integration recognition site is specific for a serine integrase. [0166] In some embodiments of the PC-derived cell or a population thereof, the second integration recognition sites is different from the at least first integration recognition attB or attP site. [0167] In some embodiments of the PC-derived cell or a population thereof, the second integration recognition site is an attB or attP site. [0168] In some embodiments of the PC-derived cell or a population thereof, the second integration ecognition sites is a modified attB or attP site. [0169] In some embodiments of the PC-derived cell or a population thereof, the second integration ecognition site is specific for BxB1 or a modified BxB1. [0170] In some embodiments of the PC-derived cell or a population thereof, the second integration recognition sites is comprised of 38 or 46 nucleotides. [0171] In some embodiments of the PC-derived cell or a population thereof, the donor polynucleotide template encodes one or more orthogonal integration recognition sites. [0172] In some embodiments of the PC-derived cell or a population thereof, a second donor polynucleotide template is integrated into the PC-derived cell genome at the second integration recognition site site-specifically incorporated into the PC-derived cell genome. [0173] In some embodiments of the PC-derived cell or a population thereof, the first donor polynucleotide template is encodes one or more therapeutic agents. [0174] In some embodiments of the PC-derived cell or a population thereof, the one or more therapeutic agents is selected from: transcription factors, receptors, signaling molecules, secreted proteins, cytokines, enzymes, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, and persistence and/or survival of the iPSCs or cells derived from the iPSC. [0175] In some embodiments of the PC-derived cell or a population thereof, the one or more therapeutic agents is a HLA class I proteins. [0176] In some embodiments of the PC-derived cell or a population thereof, the one or more therapeutic agents is a HLA class II proteins. [0177] In some embodiments of the PC-derived cell or a population thereof, the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents. [0178] In some embodiments of the PC-derived cell or a population thereof, the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agent. [0179] In some embodiments of the PC-derived cell or a population thereof, the second donor polynucleotide template comprises an inducible promoter operably linked to at least one therapeutic agent. [0180] In some embodiments of the PC-derived cell or a population thereof, the PC-derived cell or population thereof are human cells. [0181] In another aspect, this disclosure features a composition comprising any of the population of PC-derived cells described herein. [0182] In another aspect, this disclosure features a pharmaceutical composition comprising any of the population of PC-derived cells described herein and a pharmaceutically acceptable excipient or carrier. [0183] In another aspect, this disclosure features a method of treating or ameliorating or preventing a disease or condition in a subject, comprising administering a therapeutically effective amount of any of the PC-derived cells or populations described herein or any of the compositions described herein or any of the pharmaceutical composition described herein. [0184] In some embodiments of the method of treating or ameliorating or preventing a disease, the disease is a cancer. [0185] In some embodiments of the method of treating or ameliorating or preventing a disease, the disease is a muscular and/or the condition is muscle degeneration or muscle injury. [0186] In some embodiments of the method of treating or ameliorating or preventing a disease, the disease is a neuronal disease and/or the condition is neuron degeneration. [0187] In some embodiments of the method of treating or ameliorating or preventing a disease, the disease is associated with the pancreas. [0188] In another aspect, this disclosure features a method of using a pleiopluripotent cell having at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome, the method comprising: integrating a first donor polynucleotide template into the pleiopluripotent cell genome by introducing into the pleiopluripotent cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the at least one incorporated genomic integration recognition sites by the integrase. [0189] In some embodiments of the method of using the pleiopluripotent cell, the method further comprises selecting the pleiopluripotent cells having the first donor polynucleotide template site- specifically integrated into the genome. [0190] In some embodiments of the method of using the pleiopluripotent cell, the method further comprises expanding the pleiopluripotent cells in a de-differentiated state. [0191] In some embodiments of the method of using the pleiopluripotent cell, the method further comprises cryopreserving the pleiopluripotent cells. [0192] In some embodiments of the method of using the pleiopluripotent cell, the method further comprises directing differentiation of the modified pleiopluripotent cell to a hematopoietic cell. [0193] In some embodiments of the method of using the pleiopluripotent cell, the hematopoietic cell is selected from: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells. [0194] In some embodiments of the method of using the pleiopluripotent cell, the method further comprises directing differentiation of the modified pleiopluripotent cell to a neuronal cell. [0195] In some embodiments of the method of using the pleiopluripotent cell, the neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron. [0196] In some embodiments of the method of using the pleiopluripotent cell, the method further comprises directing differentiation of the modified pleiopluripotent cell to a cardiac cell. [0197] In some embodiments of the method of using the pleiopluripotent cell, the cardiac cell is selected from: a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte. [0198] In some embodiments of the method of using the pleiopluripotent cell, the method further comprises directing differentiation of the modified pleiopluripotent cell to a pancreatic cell. [0199] In some embodiments of the method of using the pleiopluripotent cell, the cardiac cell is selected from: pancreatic progenitor cell, pancreatic endoderm, an endocrine pancreatic cell, an exocrine pancreatic cell, an islet progenitor, and a beta cell. [0200] In some embodiments of the method of using the pleiopluripotent cell, the method further comprises administering the PC-derived cells or a population thereof to a patient in need thereof. 5. BRIEF DESCRIPTION OF THE DRAWINGS [0201] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where: [0202] FIG.1A-1E shows analysis of AttP variants. FIG.1A shows a non-limiting schematic of AttP mutations tested for improving integration efficiency (SEQ ID NOS: 394 and 540-542, respectively, in order of appearance). FIG. 1B shows integration efficiencies of wildtype and mutant AttP sites across a panel of AttB lengths. FIG. 1C shows a non-limiting schematic of multiplexed integration of different cargo sets at specific genomic loci. Three fluorescent cargos (GFP, mCherry, and YFP) are inserted orthogonally at three different loci (ACTB, LMNB1, NOLC1) for in-frame gene tagging. FIG.1D shows orthogonality of top 4 AttB/AttP dinucleotide pairs evaluated for GFP integration with PASTE at the ACTB locus. FIG.1E shows efficiency of multiplexed PASTE insertion of combinations of fluorophores at ACTB, LMNB1, and NOLC1 loci. Data are mean (n= 3) ± s.e.m. [0203] FIG.2 illustrates a schematic of a non-limiting example of engineered pleiopluripotent cell having a knockout of HLA Class I and/or II, one or more exogenous polypeptides capable of modulating an immune response, one or more inducible suicide genes, one or more adaptors, and optionally one or more beacons for inserting additional therapeutic genes. [0204] FIG. 3A illustrates a schematic of a donor polynucleotide template: NANOG- FKBPCasp9-ACTB-mFRBFKBPCasp9-CAG-CD47-PDL1-HLAE-3AttB and integration of the donor polynucleotide into exon 1 of the B2M locus. Abbreviations (top panel): B2M – beta-2 microglobulin. Nanog – endogenous promoter that drives expression of Nanog. FKBPCasp9: FK506-binding protein fused to caspase 9. ACTB-mFRB-FKBP-Casp9: Actin promoter driving expression of an FRB domain and FKBP-Casp9. ALFA – epitope tag. CD47: Cluster of Differentiation 47 (i.e., an example exogenous polypeptide capable of modulating an immune response). PD-L1: Programmed death-ligand 1. B2M: Beta-2-Microglobulin. HLA-E: Major Histocompatibility Complex, Class I, E. AttB1 (AA): AttB1 site with a AA dinucleotide. AttB2 (CG): AttB site with a CG dinucleotide. AttB3 (CC): AttB site with a CC dinucleotide. [0205] FIG. 3B illustrates a schematic of a donor polynucleotide template: PTRE3G-B2M- pA-hPGKprm-Tet3G-pA and integration of the donor polynucleotide template into exon 2 of the CIITA locus. Abbreviations (bottom panel): PTRE3G – TetO promoter. B2M - beta-2 microglobulin. pA – SV40 poly A tail. hPGK prm – human phosphoglycerate kinase gene promoter. Tet3G – tet transactivator. [0206] FIG.4 illustrates a schematic of an example inducible suicide gene. Left panel shows non-limiting examples Caspase9-based suicide genes. Right panel shows non-limiting examples of exogenous molecules that control activity of Caspase 9. [0207] FIG. 5 illustrates a schematic of single atgRNA and dual atgRNA approaches for beacon placement. [0208] FIG.6 shows the source of protospacer sequences for each atgRNA targeting human B2M and human CIITA genes and the dual atgRNA approach for targeting the B2M and CIITA loci. [0209] FIGs. 7A-7B illustrates designing of atgRNAs capable of beacon placement into a target genome site. Exemplary 38 or 46 base attB insertion sites are shown. FIG.7A shows a non- limiting example workflow for designing single atgRNA and dual atgRNA approaches for beacon placement. FIG.7B shows a non-limiting example workflow for designing each atgRNA. [0210] FIG.8 illustrates a non-limiting example workflow for editing pleiopluripotent cells. [0211] FIG.9 illustrates the modified atgRNA sequences for targeting the B2M and CIITA loci. Non-modified RNA are entered as ‘r_’. For example, as rA or rU.2’ O-methyl RNA are entered as ‘m_’. Phosphorothioated RNA are entered as ‘r_*’. Phosphorothioated 2’ O-methyl RNA are entered as ‘m_*’. [0212] FIGs. 10A-10B illustrate beacon placement in two different iPSC lines (iPSC line 1 was in FIG. 10A and iPSC line 2 was in FIG. 10B) across tested conditions for two different genomic loci in each iPSC line (B2M and CIITA loci). Beacon placement was determined by ddPCR and amp-seq. [0213] FIGs.11A-11B illustrate concurrent beacon placement at B2M and CIITA loci using a dual atgRNA approach for beacon placement at each loci. Beacon placement was determined by ddPCR. FIG.11A shows raw data from ddPCR. FIG.11B shows a histogram summarizing data from FIG.11A. [0214] FIGs. 12A-12B illustrate beacon placement data for various iPSC lines. FIG. 12A shows raw ddPCR data for beacon placement at the B2M loci and the CIITA loci for 22 iPSC lines. FIG.12B shows a histogram summarizing ddPCR data for three iPSC lines. Lines 1 and 9 show 100% (i.e., bi-allelic) beacon placement at both B2M and CIITA loci. [0215] FIG.13 shows Amp-seq data for beacon placement in six iPSC cell lines generated in Example 2. [0216] FIGs.14A-14B illustrate a schematic of the digital droplet PCR (ddPCR) assay used to detect integration of a donor polynucleotide at the B2M locus (FIG. 14A) or the CIITA locus (FIG.14B). Similar designs can be used to determine integration at other genomic loci. [0217] FIG. 15 shows data for integration of a 6 kilobase (kb) first donor polynucleotide template into the B2M locus (site 1) and a 5kb second donor polynucleotide template into the CIITA locus (site 2). Data is presented as transductions that include a single donor polynucleotide template, thereby measuring a single integration of donor polynucleotide template at either the B2M locus or the CIITA locus (single integration), referred to in FIG.15 as “single.” Data is also presented for transductions that include both the first and second donor polynucleotide templates, thereby showing concurrent integration of the donor polynucleotide templates at the B2M locus and CIITA locus, referred to in FIG.15 as “Duplex”. [0218] FIG.16 shows data for programmable gene insertion (i.e., integration) of a 31 kb donor polynucleotide template at the CIITA locus that has an integration recognition site site-specifically incorporated into exon 2. The 31 kb donor polynucleotide (Adv donor) was delivered to the induced pluripotent stem cell (iPSC) using adenovirus at MOIs of 0.01, 0.1, 1, and 10. [0219] FIG.17 shows polynucleotide and amino acid sequences for the donor polynucleotide template described in FIG.3A. FIG.17 includes the polynucleotide sequence (SEQ ID NO: 616) of the donor polynucleotide template that is integrated into the B2M locus. FIG.17 also includes the FKBP-linker-Caspase9 amino acid sequence (SEQ ID NO: 662); the mFRB-linker-FKBP- Caspase9-P2A-DeltaTK amino acid sequence (SEQ ID NO: 663); and the alpha tag-CD47-P2A- PDL1-T2A-B2M-HLA-E amino acid sequence (SEQ ID NO: 664). [0220] FIG. 18 shows polynucleotide and amino acid sequences for donor polynucleotide template described in FIG.3B. FIG.18 includes the polynucleotide sequence (SEQ ID NO: 617) of the donor polynucleotide template that is integrated into the CIITA locus. FIG.18 also includes the B2M amino acid sequence (SEQ ID NO: 665) and the Tet3G amino acid sequence (SEQ ID NO: 666). [0221] FIG.19A shows ddPCR data for percent beacon placement in the CD52 locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis and identified in the key. [0222] FIG.19B shows next-generation sequencing data for percent beacon placement in the CD52 locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis that include a subset of the atgRNAs identified in the key in FIG.19A. [0223] FIG.20A shows ddPCR data for percent beacon placement in the CISH locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis, which are also described in Example 6. [0224] FIG.20B shows next-generation sequencing data for percent beacon placement in the CISH locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis. The pairs in FIG.20B correspond to the pairs in FIG.20A as follows: pair 1 corresponds to SP01-N01, pair 2 corresponds to SP02-N02, pair 5 corresponds to SP05-N04, pair 3 corresponds to SP03-N03, and pair 4 corresponds to SP04-N04. [0225] FIG. 21A shows ddPCR data for percent beacon placement in the ADRA2A locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis, which are also described in Example 7. [0226] FIG.21B shows next-generation sequencing data for percent beacon placement in the ADRA2A locus in iPSC clone #17 following electroporation with the atgRNA pairs indicated on the x-axis. The pairs in FIG. 21B correspond to the pairs in FIG. 21A as follows: pair 1 corresponds to SP01-N01, pair 2 corresponds to SP02-N02, pair 3 corresponds to SP03-N03, pair 4 corresponds to SP04-N04, and pair 5 corresponds to SP05-N05. [0227] FIG. 22 shows ddPCR data for percent beacon placement in the B2M, CIITA, and CD52 loci in a wild type iPSC line following electroporation with the indicated combinations of atgRNA pairs. Abbreviations: “3X” indicates electroporation with three atgRNA pairs for multiplex beacon placement; bmp001, bmp002, bmp003, and mod2 indicate the atgRNA is modified. [0228] FIG.23 shows ddPCR data for percent beacon placement in the TRAC and AAVS1 loci in iPSC clone # 17 following electroporation with the atgRNA pairs indicated on the x-axis. Data is shown for negative control, electroporation of TRAC atgRNA pair only, electroporation of AAVS1 atgRNA pair only, and the co-electroporation of the TRAC and AAVS1 atgRNA pairs. DKO #17 indicates that iPSC clone includes beacons site-specifically incorporated into B2M and CIITA loci prior to being electroporated with the TRAC and AAVS1 atgRNAs. [0229] FIG.24 shows a non-limiting example of the clonal isolation process for generating an iPSC clone. The overall process takes roughly a month from electroporation to NGS analysis. First, electroporation (EP) is used to place beacons in the iPS cells. Electroporated iPSCs are grown for 72 hours and part of the iPSC culture is harvested for genomic DNA to get an initial read of beacon placement. If cells are positive for beacon placement, electroporation is scaled up from a 48 well plate to a 24 well plate and then eventually to 6 wells. A confluent 6 well population of cells is diluted such that a single cell is placed in each well of a 96-well plate across multiple 96 well plates. These cells take roughly two weeks to reach the point of needing to be harvested. Cell growth is monitored and only clones with good morphology and growth rates are expanded and/or harvested for a second round of ddPCR. Cells with high performing beacon placement (e.g., beacon placement at both the TRAC and AAVS1 loci) are scaled up a final time to 6 well plates. The last step being to send samples for NGS analysis and cryopreserve the remaining cells. [0230] FIG.25 shows next generation sequencing data from iPSC clones generated using the method described in FIG.24. Abbreviations: Total # = total number of reads; and merge R1/R2 = merged reads 1 and 2. [0231] FIG. 26 shows ddPCR data for programmable gene insertion of four different donor polynucleotide templates at the B2M, CIITA, TRAC, and AAVS1 loci in iPSC clones #16 and #29. Each donor polynucleotide template includes an AttP site having a different central dinucleotide that enables integration at its corresponding (cognate) AttB site that has been site-specifically integrated into the iPS cell genome at one of the indicated loci. Controls include no BxB1 control (i.e., donor polynucleotide templates but no BxB1) and a no donor polynucleotide template control (i.e., a BxB1 polypeptide or polynucleotide encoding a BxB1 polypeptide but no donor polynucleotide templates. [0232] FIG. 27 shows ddPCR data for programmable gene insertion of four different donor polynucleotide templates at the B2M, CIITA, TRAC, and AAVS1 loci in iPSC clones #30. Each donor polynucleotide template includes an AttP site having a different central dinucleotide that enables integration at its corresponding (cognate) AttB site that has been site-specifically integrated into the iPS cell genome at one of the indicated loci. 6. DETAILED DESCRIPTION OF THE INVENTION [0233] In one aspect, the methods described herein enable multiplexed programmable gene insertion whereby multiple edits (e.g., beacon placement, donor polynucleotide incorporation, or a combination thereof) can be performed sequentially or simultaneously in a single pleiopluripotent cell, thereby reducing the time and cost associated with conventional methods of editing pleiopluripotent cells. Additionally, the methods described herein avoid the need for inducing double strand breaks, which increases accuracy of genome integration and reduces likelihood of off-target events. For example, using a beacon (e.g., an integration recognition site) incorporated at a particular genomic location to guide integration of the donor polynucleotide template increases integration efficiency of the donor polynucleotide template. Moreover, the methods described herein enable integration of larger donor polynucleotide template sequences compared to conventional methods which have payload capacity limitations. This is enabled, at least in part, by using single atgRNA (and optionally a nicking gRNA) or dual atgRNA to guide precise beacon placement (i.e., incorporation of one or more integration recognition sites) at desired loci. One application of these methods is the generation of pleiopluripotent cells that include one or more of (i) knockout of HLA Class I and/or Class II molecules, (ii) expression of self-molecules (e.g., one or more exogenous polypeptides capable of modulating an immune response), (iii) expression one or more controllable (e.g., inducible) suicide genes, (iv) expression of one or more adaptors (e.g., a tag), and (v) one or more landing pads (e.g., beacons) incorporated into the genome for integrating additional therapeutic agents (see FIG.2). Cells comprising one or more of these features provide a source of cells suitable for differentiation to a cell type of interest (e.g., without limitation, a hematopoietic cell, a neuronal cell, a cardiac cell, and a pancreatic cell) and use in cell therapy (e.g., allogeneic cell therapy). 6.1. Terminology [0234] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. As used herein, the following terms have the meanings ascribed to them below. [0235] “Gene editor” as used herein, is a protein that that can be used to perform gene editing, gene modification, gene insertion, gene deletion, or gene inversion. As used herein, the terms “gene editor polynucleotide” refers to a polynucleotide sequence encoding the gene editor polypeptide. Such an enzyme or enzyme fusion may contain DNA or RNA targetable nuclease protein (i.e., Cas protein, ADAR, or ADAT), wherein target specificity is mediated by a complexed nucleic acid (i.e., guide RNA). Such an enzyme or enzyme fusion may be a DNA/RNA targetable protein, wherein target specificity is mediated by internal, conjugated, fused, or linked amino acids, such as within TALENs, ZFNs, or meganucleases. The skilled person in the art would appreciate that the gene editor can demonstrate targeted nuclease activity, targeted binding with no nuclease activity, or targeted nickase activity (or cleavase activity). A gene editor comprising a targetable protein may be fused or linked to one or more proteins or protein fragment motifs. Gene editors may be fused, linked, complexed, operate in cis or trans to one or more integrase, recombinase, polymerase, telomerase, reverse transcriptase, or invertase. A gene editor can be a prime editor fusion protein or a gene writer fusion protein. [0236] “Prime editor fusion protein” as used herein, describes a protein that is used in prime editing. “Prime editor system” as used herein describes the components used in prime editing. Prime editing uses CRISPR enzyme that nicks or cuts only single strand of double stranded DNA, i.e., a nickase; the nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts. Such an enzyme can be a catalytically impaired Cas9 endonuclease (a nickase). Such an enzyme can be a Casl2a/b, MAD7, or variant thereof. The nickase is fused to an engineered reverse transcriptase (RT). The nickase is programmed (directed) with an attachment site-containing guide RNA (or a prime-editing guide RNA (pegRNA)). The skilled person in the art would appreciate that the pegRNA both specifies the target site and encodes the desired edit. Described herein, are attachment site-containing guide RNA (atgRNA) that both specifies the target and encodes for the desired integrase target recognition site. The nickase may be programmed (directed) with an atgRNA. Advantageously the nickase is a catalytically-impaired Cas9 endonuclease, a Cas9 nickase, that is fused to the reverse transcriptase. During genetic editing, the Cas9 nickase part of the protein is guided to the DNA target site by the atgRNA (or pegRNA), whereby a nick or single stranded cut occurs. The reverse transcriptase domain then uses the atgRNA (or pegRNA) to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Afterward, optionally, the prime editor (PE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA). Other enzymes that can be used to nick or cut only a single strand of double stranded DNA includes a cleavase (e.g., cleavase I enzyme). [0237] In some embodiments, an additional agent or agents may be added that improve the efficiency and outcome purity of the prime edit. In some embodiments, the agent may be chemical or biological and disrupt DNA mismatch repair (MMR) processes at or near the edit site (i.e., PE4 and PE5 and PEmax architecture by Chen et al. Cell, 184, 1-18, October 28, 2021; Chen et al. is incorporated herein by reference). In typical embodiments, the agent is a MMR-inhibiting protein. In certain embodiments, the MMR-inhibiting protein is dominant negative MMR protein. In certain embodiments, the dominant negative MMR protein is MLH1dn. In particular embodiments, the MMR-inhibiting agent is incorporated into the single nucleic acid construct design described herein. In some embodiments, the MMR-inhibiting agent is linked or fused to the prime editor protein fusion, which may or may not have a linked or fused integrase. In some embodiments, the MMR-inhibiting agent is linked or fused to the Gene Writer™ protein, which may or may not have a linked or fused integrase. [0238] The prime editor or gene editor system can be used to achieve DNA deletion and replacement. In some embodiments, the DNA deletion replacement is induced using a pair of atgRNAs (or pegRNA) that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair (i.e., PrimeDel by Choi et al. Nat. Biotechnology, October 14, 2021; Choi et al. is incorporated herein by reference and TwinPE by Anzalone et al., Nat. Biotech. 40: 731–740 (2022); Anzalone et al. is incorporated herein by reference). In some embodiments described herein, the DNA deletion is induced using a single atgRNA. In some embodiments, the DNA deletion and replacement is induced using a wild type Cas9 prime editor (PE-Cas9) system (i.e., PEDAR by Jiang et al. Nat. Biotechnology, October 14, 2021; Jiang et al. is incorporated herein by reference). In some embodiments, the DNA replacement is an integrase target recognition site or recombinase target recognition site. In certain embodiments, the constructs and methods described herein may be utilized to incorporate the pair of pegRNAs used in PrimeDel, TwinPE (WO2021226558, which is hereby incorporated by reference herein in its entirety), or PEDAR, the prime editor fusion protein or Gene Writer protein, optionally a nickase guide RNA (ngRNA), an integrase, a nucleic acid cargo, and optionally a recombinase into a single nucleic acid construct described herein. The integrase may be directly linked, for example by a peptide linker, to the prime editor fusion or gene writer protein. [0239] In some embodiments, the prime editors can refer to a retrovirus or lentivirus reverse transcriptase such as a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (RT) fused to a CRISPR enzyme nickase such as a Cas9 H840A nickase, a Cas9nickase. In some embodiments, the prime editors can refer to a retrovirus or lentivirus reverse transcriptase such as a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (RT) fused to a cleavase. In some embodiments the RT can be fused at, near or to the C-terminus of a Cas9nickase, e.g., Cas9 H840A. Fusing the RT to the C-terminus region, e.g., to the C-terminus, of the Cas9 nickase may result in higher editing efficiency. Such a complex is called PEI. In some embodiments, the CRISPR enzyme nickase, e.g., Cas9(H840A), i.e., a Cas9nickase, can be linked to a non-M-MLV reverse transcriptase such as an AMV-RT or XRT (Cas9(H840A)-AMV-RT or XRT). In some embodiments, instead of the CRISPR enzyme nickase being a Cas9 (H840A), i.e., instead of being a Cas9 nickase, the CRISPR enzyme nickase instead can be a CRISPR enzyme that naturally is a nickase or cuts a single strand of double stranded DNA; for instance, the CRISPR enzyme nickase can be Casl2a/b. Alternatively, the CRISPR enzyme nickase can be another mutation of Cas9, such as Cas9(Dl0A). A CRISPR enzyme, such as a CRISPR enzyme nickase, such as Cas9 (wild type), Cas9(H840A), Cas9(Dl0A) or Cas 12a/b nickase can be fused in some embodiments to a pentamutant of M-MLV RT (D200N/ L603W/ T330P/ T306K/ W313F), whereby there can be up to about 45-fold higher efficiency, and this is called PE2. In some embodiments, the M-MLV RT comprise one or more of the mutations Y8H, P51L, S56A, S67R, E69K, Vl29P, L139P, Tl97A, H204R, V223H, T246E, N249D, E286R, Q2911, E302K, E302R, F309N, M320L, P330E, L435G, L435R, N454K, D524A, D524G, D524N, E562Q, D583N, H594Q, E607K, D653N, and L671P. Specific M-MLV RT mutations are shown in Table 1.
Figure imgf000035_0001
[0240] In some embodiments, the reverse transcriptase can also be a wild-type or modified transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV RT), Feline Immunodeficiency Virus reverse transcriptase (FIV-RT), FeLV-RT (Feline leukemia virus reverse transcriptase), HIV-RT (Human Immunodeficiency Virus reverse transcriptase). In some embodiments, the reverse transcriptase can be a fusion of MMuLV to the Sto7d DNA binding domain (see Ionnidi et al.; https://doi.org/10.1101/2021.11.01.466786). The fusion of MMuLV to the Sto7d DNA binding domain sequence is given in Table 2.
Figure imgf000036_0001
[0241] PE3, PE3b, PE4, PE5, and/or PEmax, which a skilled person can incorporate into the gene editor polypeptide, involves nicking the non-edited strand, potentially causing the cell to remake that strand using the edited strand as the template to induce HR. The nicking of the non-edited strand can involve the use of a nicking guide RNA (ngRNA). [0242] The skilled person can readily incorporate into a gene editor polypeptide described herein a prime editing or CRISPR system. Examples of prime editors can be found in the following: WO2020/191153, WO2020/191171, WO2020/191233, WO2020/191234, WO2020/191239, WO2020/191241, WO2020/191242, WO2020/191243, WO2020/191245, WO2020/191246, WO2020/191248, WO2020/191249, each of which is incorporated by reference herein in its entirety. In addition, mention is made, and can be used herein, of CRISPR Patent Applications and Patents of the Zhang laboratory and/or Broad Institute, Inc. and Massachusetts Institute of Technology and/or Broad Institute, Inc., Massachusetts Institute of Technology and President and Fellows of Harvard College and/or Editas Medicine, Inc. Broad Institute, Inc., The University of Iowa Research Foundation and Massachusetts Institute of Technology, including those claiming priority to US Application 61/736,527, filed December 12, 2012, including US Patents 11,104,937, 11,091,798, 11,060,115, 11,041,173, 11,021,740, 11,008,588, 11,001,829, 10,968,257, 10,954,514, 10,946,108, 10,930,367, 10,876,100, 10,851,357, 10,781,444, 10,711,285, 10,689,691, 10,648,020, 10,640,788, 10,577,630, 10,550,372, 10,494,621, 10,377,998, 10,266,887, 10,266,886, 10,190,137, 9,840,713, 9,822,372, 9,790,490, 8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616, 8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945, and 8,697,359; CRISPR Patent Applications and Patents of the Doudna laboratory and/or of Regents of the University of California, the University of Vienna and Emmanuelle Charpentier, including those claiming priority to US application 61/652,086, filed May 25, 2012, and/or 61/716,256, filed October 19, 2012, and/or 61/757,640, filed January 28, 2013, and/or 61/765,576, filed February 15, 2013 and/or 13/842,859, including US Patents 11,028,412, 11,008,590, 11,008,589, 11,001,863, 10,988,782, 10,988,780, 10,982,231, 10,982,230, 10,900,054, 10,793,878, 10,774,344, 10,752,920, 10,676,759, 10,669,560, 10,640,791, 10,626,419, 10,612,045, 10,597,680, 10,577,631, 10,570,419, 10,563,227, 10,550,407, 10,533,190, 10,526,619, 10,519,467, 10,513,712, 10,487,341, 10,443,076, 10,428,352, 10,421,980, 10,415,061, 10,407,697, 10,400,253, 10,385,360, 10,358,659, 10,358,658, 10,351,878, 10,337,029, 10,308,961, 10,301,651, 10,266,850, 10,227,611, 10,113,167, and 10,000,772; CRISPR Patent Applications and Patents of Vilnius University and/or the Siksnys laboratory, including those claiming priority to US application 62/046384 and/or 61/625,420 and/or 61/613,373 and/or PCT/IB2015/056756, including US Patent 10,385,336; CRISPR Patent Applications and Patents of the President and Fellows of Harvard College, including those of George Church’s laboratory and/or claiming priority to US application 61/738,355, filed December 17, 2012, including 11,111,521, 11,085,072, 11,064,684, 10,959,413, 10,925,263, 10,851,369, 10,787,684, 10,767,194, 10,717,990, 10,683,490, 10,640,789, 10,563,225, 10,435,708, 10,435,679, 10,375,938, 10,329,587, 10,273,501, 10,100,291, 9,970,024, 9,914,939, 9,777,262, 9,587,252, 9,267,135, 9,260,723, 9,074,199, 9,023,649; CRISPR Patent Applications and Patents of the President and Fellows of Harvard College, including those of David Liu’s laboratory, including 11,111,472, 11,104,967, 11,078,469, 11,071,790, 11,053,481, 11,046,948, 10,954,548, 10,947,530, 10,912,833, 10,858,639, 10,745,677, 10,704,062, 10,682,410, 10,612,011, 10,597,679, 10,508,298, 10,465,176, 10,323,236, 10,227,581, 10,167,457, 10,113,163, 10,077,453, 9,999,671, 9,840,699, 9,737,604, 9,526,784, 9,388,430, 9,359,599, 9,340,800, 9,340,799, 9,322,037, 9,322,006, 9,228,207, 9,163,284, and 9,068,179; and CRISPR Patent Applications and Patents of Toolgen Incorporated and/or the Kim laboratory and/or claiming priority to US application 61/717,324, filed October 23, 2012 and/or 61/803,599, filed March 20, 2013 and/or 61/837,481, filed June 20, 2013 and/or 62/033,852, filed August 6, 2014 and/or PCT/KR2013/009488 and/or PCT/KR2015/008269, including US Patent 10,851,380, and 10,519,454; and CRISPR Patent Applications and Patents of Sigma and/or Millipore and/or the Chen laboratory and/or claiming priority to US application 61/734,256, filed December 6, 2012 and/or 61/758,624, filed January 30, 2013 and/or 61/761,046, filed February 5, 2013 and/or 61/794,422, filed March 15, 2013, including US Patent 10,731,181, each of which is hereby incorporated herein by reference, and from the disclosures of the foregoing, the skilled person can readily make and use a prime editing or CRISPR system, and can especially appreciate impaired endonucleases, such as a mutated Cas9 that only nicks a single strand of DNA and is hence a nickase, or a CRISPR enzyme that only makes a single-stranded cut that can be employed in a PASTE system of the invention. Further, from the disclosures of the foregoing, the skilled person can incorporate the selected CRISPR enzyme, as part of the gene editor composition described herein. Additional gene editor polypeptides are as described in WO 2023/076898; WO 2023/015014; WO 2023/070062; WO2023288332; WO2023015318; WO2023004439; WO2023070110; WO2022256714; and WO2023283092, each of which are hereby incorporated by reference in their entireties. Additional gene editor polypeptides are as described in U.S. Patent Pub.2023/0059368, which is hereby incorporated by reference in its entirety. [0243] Prior to RT-mediated edit incorporation, the prime editor protein (1) site- specifically targets a genomic locus and (2) performs a catalytic cut or nick. These steps are typically performed by a CRISPR-Cas. However, in some embodiments the Cas protein may be substituted by other nucleic acid programmable DNA binding proteins (napDNAbp) such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or meganucleases. In addition, to the extent the “targeting rules” of other napDNAbp are known or are newly determined, it becomes possible to use new napDNAbp, beyond Cas9, to site specifically target and modify genomic sites of interest. [0244] Similar to a prime editor protein, a Gene Writer can introduce novel DNA elements, such as an integration target site, into a DNA locus. A Gene Writer protein comprises: (A) a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain, and either (x) an endonuclease domain that contains DNA binding functionality or (y) an endonuclease domain and separate DNA binding domain; and (B) a template RNA comprising (i) a sequence that binds the polypeptide and (ii) a heterologous insert sequence. Examples of such Gene Writer™ proteins and related systems can be found in US20200109398, which is incorporated by reference herein in its entirety. [0245] In some embodiments, the prime editor or Gene Writer protein fusion or prime editor protein linked or fused to an integrase is expressed as a split construct. In typical embodiments, the split construct in reconstituted in a cell. In some embodiments, the split construct can be fused or ligated via intein protein splicing. In some embodiments, the split construct can be reconstituted via protein-protein inter-molecular bonding and/or interactions. In some embodiments, the split construct can be reconstituted via chemical, biological, or environmental induced oligomerization. In some embodiments, the split construct can be reconstituted via nanobody binding ALFA-tagged proteins. In certain embodiments, the split construct can be adapted into one or more single nucleic acid polynucleotides. [0246] In some embodiments, an integrase or recombinase is directly linked or fused, for example by a peptide linker, which may be cleavable or non-cleavable, to the prime editor fusion protein (i.e., fused Cas9 nickase-reverse transcriptase) or Gene Writer protein. Suitable linkers, for example between the Cas9, RT, and integrase, may be selected from Table 3:
Figure imgf000039_0001
Table 3
Figure imgf000040_0001
6.2. Type II CRISPR proteins [0247] The skilled person can incorporate a selected CRISPR enzyme, described below, as part of the prime editor fusion, as a component of the gene editor polypeptide described herein. Streptococcus pyogenes Cas9 (SpCas9), the most common enzyme used in genome-editing applications, is a large nuclease of 1368 amino acid residues. Advantages of SpCas9 include its short, 5′-NGG-3′ PAM and very high average editing efficiency. SpCas9 consists of two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe can be divided into three regions, a long a helix referred to as the bridge helix (residues 60–93), the REC1 (residues 94–179 and 308–713) domain, and the REC2 (residues 180–307) domain. The NUC lobe consists of the RuvC (residues 1–59, 718–769, and 909–1098), HNH (residues 775–908), and PAM-interacting (PI) (residues 1099–1368) domains. The negatively charged sgRNA:target DNA heteroduplex is accommodated in a positively charged groove at the interface between the REC and NUC lobes. In the NUC lobe, the RuvC domain is assembled from the three split RuvC motifs (RuvC I–III) and interfaces with the PI domain to form a positively charged surface that interacts with the 30 tail of the sgRNA. The HNH domain lies between the RuvC II–III motifs and forms only a few contacts with the rest of the protein. Structural aspects of SpCas9 are described by Nishimasu et al., Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA, Cell 156, 935-949, February 27, 2014. [0248] REC lobe: The REC lobe includes the REC1 and REC2 domains. The REC2 domain does not contact the bound guide:target heteroduplex, indicating that truncation of REC lobe may be tolerated by SpCas9. Further, SpCas9 mutant lacking the REC2 domain (D175–307) retained ~50% of the wild-type Cas9 activity, indicating that the REC2 domain is not critical for DNA cleavage. In striking contrast, the deletion of either the repeat-interacting region (D97–150) or the anti-repeat-interacting region (D312–409) of the REC1 domain abolished the DNA cleavage activity, indicating that the recognition of the repeat:anti-repeat duplex by the REC1 domain is critical for the Cas9 function. [0249] PAM-Interacting domain: The NUC lobe contains the PAM-interacting (PI) domain that is positioned to recognize the PAM sequence on the noncomplementary DNA strand. The PI domain of SpCas9 is required for the recognition of 5’-NGG-3’ PAM, and deletion of the PI domain (Δ1099-1368) abolished the cleavage activity, indicating that the PI domain is critical for SpCas9 function and a major determinant for the PAM specificity. [0250] RuvC domain: The RuvC nucleases of SpCas9 have an RNase H fold and four catalytic residues, Asp10 (Ala), Glu762, His983, and Asp986, that are critical for the two-metal cleavage of the noncomplementary strand of the target DNA. In addition to the conserved RNase H fold, the Cas9 RuvC domain has other structural elements involved in interactions with the guide:target heteroduplex (an end-capping loop between α42 and α43) and the PI domain/stem loop 3 (β hairpin formed by β3 and β4). [0251] HNH domain: SpCas9 HNH nucleases have three catalytic residues, Asp839, His840, and Asn863 and cleave the complementary strand of the target DNA through a single-metal mechanism. [0252] sgRNA:DNA recognition: The sgRNA guide region is primarily recognized by the REC lobe. The backbone phosphate groups of the guide region (nucleotides 2, 4–6, and 13–20) interact with the REC1 domain (Arg165, Gly166, Arg403, Asn407, Lys510, Tyr515, and Arg661) and the bridge helix (Arg63, Arg66, Arg70, Arg71, Arg74, and Arg78). The 20-hydroxyl groups of G1, C15, U16, and G19 hydrogen bond with Val1009, Tyr450, Arg447/Ile448, and Thr404, respectively. [0253] A mutational analysis demonstrated that the R66A, R70A, and R74A mutations on the bridge helix markedly reduced the DNA cleavage activities, highlighting the functional significance of the recognition of the sgRNA ‘‘seed’’ region by the bridge helix. Although Arg78 and Arg165 also interact with the ‘‘seed’’ region, the R78A and R165A mutants showed only moderately decreased activities. These results are consistent with the fact that Arg66, Arg70, and Arg74 form multiple salt bridges with the sgRNA backbone, whereas Arg78 and Arg165 form a single salt bridge with the sgRNA backbone. Moreover, the alanine mutations of the repeat:anti- repeat duplex-interacting residues (Arg75 and Lys163) and the stemloop-1-interacting residue (Arg69) resulted in decreased DNA cleavage activity, confirming the functional importance of the recognition of the repeat:anti-repeat duplex and stem loop 1 by Cas9. [0254] RNA-guided DNA targeting: SpCas9 recognizes the guide:target heteroduplex in a sequence-independent manner. The backbone phosphate groups of the target DNA (nucleotides 1, 9–11, 13, and 20) interact with the REC1 (Asn497, Trp659, Arg661, and Gln695), RuvC (Gln926), and PI (Glu1108) domains. The C2’ atoms of the target DNA (nucleotides 5, 7, 8, 11, 19, and 20) form van der Waals interactions with the REC1 domain (Leu169, Tyr450, Met495, Met694, and His698) and the RuvC domain (Ala728). The terminal base pair of the guide:target heteroduplex (G1:C20’) is recognized by the RuvC domain via end-capping interactions; the sgRNA G1 and target DNA C20’ nucleobases interact with the Tyr1013 and Val1015 side chains, respectively, whereas the 20-hydroxyl and phosphate groups of sgRNA G1 interact with Val1009 and Gln926, respectively. [0255] Repeat:Anti-Repeat duplex recognition: The nucleobases of U23/A49 and A42/G43 hydrogen bond with the side chain of Arg1122 and the main-chain carbonyl group of Phe351, respectively. The nucleobase of the flipped U44 is sandwiched between Tyr325 and His328, with its N3 atom hydrogen bonded with Tyr325, whereas the nucleobase of the unpaired G43 stacks with Tyr359 and hydrogen bonds with Asp364. [0256] The nucleobases of G21 and U50 in the G21:U50 wobble pair stack with the terminal C20:G10 pair in the guide:target heteroduplex and Tyr72 on the bridge helix, respectively, with the U50 O4 atom hydrogen bonded with Arg75. Notably, A51 adopts the syn conformation and is oriented in the direction opposite to U50. The nucleobase of A51 is sandwiched between Phe1105 and U63, with its N1, N6, and N7 atoms hydrogen bonded with G62, Gly1103, and Phe1105, respectively. [0257] Stem-loop recognition: Stem loop 1 is primarily recognized by the REC lobe, together with the PI domain. The backbone phosphate groups of stem loop 1 (nucleotides 52, 53, and 59– 61) interact with the REC1 domain (Leu455, Ser460, Arg467, Thr472, and Ile473), the PI domain (Lys1123 and Lys1124), and the bridge helix (Arg70 and Arg74), with the 20-hydroxyl group of G58 hydrogen bonded with Leu455. A52 interacts with Phe1105 through a face-to-edge p-p stacking interaction, and the flipped U59 nucleobase hydrogen bonds with Asn77. [0258] The single-stranded linker and stem loops 2 and 3 are primarily recognized by the NUC lobe. The backbone phosphate groups of the linker (nucleotides 63–65 and 67) interact with the RuvC domain (Glu57, Lys742, and Lys1097), the PI domain (Thr1102), and the bridge helix (Arg69), with the 20-hydroxyl groups of U64 and A65 hydrogen bonded with Glu57 and His721, respectively. The C67 nucleobase forms two hydrogen bonds with Val1100. [0259] Stem loop 2 is recognized by Cas9 via the interactions between the NUC lobe and the non-Watson-Crick A68:G81 pair, which is formed by direct (between the A68 N6 and G81 O6 atoms) and water-mediated (between the A68 N1 and G81 N1 atoms) hydrogen-bonding interactions. The A68 and G81 nucleobases contact Ser1351 and Tyr1356, respectively, whereas the A68:G81 pair interacts with Thr1358 via a water-mediated hydrogen bond. The 20-hydroxyl group of A68 hydrogen bonds with His1349, whereas the G81 nucleobase hydrogen bonds with Lys33. [0260] Stem loop 3 interacts with the NUC lobe more extensively, as compared to stem loop 2. The backbone phosphate group of G92 interacts with the RuvC domain (Arg40 and Lys44), whereas the G89 and U90 nucleobases hydrogen bond with Gln1272 and Glu1225/Ala1227, respectively. The A88 and C91 nucleobases are recognized by Asn46 via multiple hydrogen- bonding interactions. [0261] Cas9 proteins smaller than SpCas9 allow more efficient packaging of nucleic acids encoding CRISPR systems, e.g., Cas9 and sgRNA into one rAAV (“all-in-one-AAV”) particle. In addition, efficient packaging of CRISPR systems can be achieved in other viral vector systems (i.e., lentiviral, hd-AAV, etc.) and non viral vector systems (i.e., lipid nanoparticle). Small Cas9 proteins can be advantageous for multidomain-Cas-nuclease-based systems for prime editing. Well characterized smaller Cas9 proteins include Staphylococcus aureus (SauCas9, 1053 amino acid residues) and Campylobacter jejuni (CjCas9, 984 amino residues). However, both recognize longer PAMs, 5′-NNGRRT-3′ for SauCas9 (R = A or G) and 5′-NNNNRYAC-3′ for CjCas9 (Y = C or T), which reduces the number of uniquely addressable target sites in the genome, in comparison to the NGG SpCas9 PAM. Among smaller Cas9s, Schmidt et al. identified Staphylococcus lugdunensis (Slu) Cas9 as having genome-editing activity and provided homology mapping to SpCas9 and SauCas9 to facilitate generation of nickases and inactive (“dead”) enzymes (Schmidt et al., 2021, Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases. Nat Commun 12, 4219. doi.org/10.1038/s41467-021- 24454-5) and engineered nucleases with higher cleavage activity by fragmenting and shuffling Cas9 DNAs. The small Cas9s and nickases are useful in the instant disclosure. [0262] Besides dead Cas9 and Cas9 nickase variants, the Cas9 proteins used herein may also include other “Cas9 variants” having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9. In some embodiments, the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9. [0263] In some embodiments, the disclosure also may utilize Cas9 fragments that retain their functionality and that are fragments of any herein disclosed Cas9 protein. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. [0264] In various embodiments, the prime editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 variants. 6.2.1. Table 4. Cas9 Orthologs.
Figure imgf000045_0001
Figure imgf000046_0001
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[0265] In some embodiments, prime editors utilized herein comprise CRISPR-Cas system enzymes other than type II enzymes. In certain embodiments, prime editors comprise type V or type VI CRISPR-Cas system enzymes. It will be appreciated that certain CRISPR enzymes exhibit promiscuous ssDNA cleavage activity and appropriate precautions should be considered. In certain embodiments, prime editors comprise a nickase or a dead CRISPR with nuclease function comprised in a different component. [0266] In various embodiments, the nucleic acid programmable DNA binding proteins utilized herein include, without limitation, Cas9 (e.g., dCas9 and nCas9), Cas12a (Cpf1), Cas12b1 (C2c1), Cas12b2, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), C2c4, C2c5, C2c8, C2c9, C2c10, Cas13a (C2c2), Cas13b (C2c6), Cas13c (C2c7), Cas13d, and Argonaute. Cas-equivalents further include those described in Makarova et al., “C2c2 is a single-component programmable RNA- guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) and Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol.1. No.5, 2018, the contents of which are incorporated herein by reference. One example of a nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (i.e, Cas12a (Cpf1)). Similar to Cas9, Cas12a (Cpf1) is also a Class 2 CRISPR effector, but it is a member of type V subgroup of enzymes, rather than the type II subgroup. It has been shown that Cas12a (Cpf1) mediates robust DNA interference with features distinct from Cas9. Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T- rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p.949-962; the entire contents of which is hereby incorporated by reference. 6.3. Type V CRISPR proteins [0267] In some embodiments, prime editors used herein comprise the type V CRISPR family includes Francisella novicida U112 Cpf1 (FnCpf1) also known as FnCas12a. FnCpf1 adopts a bilobed architecture with the two lobes connected by the wedge (WED) domain. The N-terminal REC lobe consists of two a-helical domains (REC1 and REC2) that have been shown to coordinate the crRNA-target DNA heteroduplex. The C-terminal NUC lobe consists of the C-terminal RuvC and Nuc domains involved in target cleavage, the arginine-rich bridge helix (BH), and the PAM- interacting (PI) domain. The repeat-derived segment of the crRNA forms a pseudoknot stabilized by intra-molecular base-pairing and hydrogen-bonding interactions. The pseudoknot is coordinated by residues from the WED, RuvC, and REC2 domains, as well as by two hydrated magnesium cations. Notably, nucleotides 1–5 of the crRNA are ordered in the central cavity of FnCas12a and adopt an A-form-like helical conformation. Conformational ordering of the seed sequence is facilitated by multiple interactions between the ribose and phosphate moieties of the crRNA backbone and FnCpf1 residues in the WED and REC1 domains. These include residues Thr16, Lys595, His804, and His881 from the WED domain and residues Tyr47, Lys51, Phe182, and Arg186 from the REC1 domain. The structure of the FnCas12a-crRNA complex further reveals that the bases of the seed sequence are solvent exposed and poised for hybridization with target DNA. Structural aspects of FnCpf1 are described by Swarts et al., Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a, Molecular Cell 66, 221-233, April 20, 2017. [0268] Pre-crRNA processing: Essential residues for crRNA processing include His843, Lys852, and Lys869. Structural observations are consistent with an acid-base catalytic mechanism in which Lys869 acts as the general base catalyst to deprotonate the attacking 2’-hydroxyl group of U(-19), while His843 acts as a general acid to protonate the 5’-oxygen leaving group of A(-18). In turn, the side chain of Lys852 is involved in charge stabilization of the transition state. Collectively, these interactions facilitate the intra-molecular attack of the 20-hydroxyl group of U(-19) on the scissile phosphate and promote the formation of the 2’,3’-cyclic phosphate product. [0269] R-loop formation: The crRNA-target DNA strand heteroduplex is enclosed in the central cavity formed by the REC and NUC lobes and interacts extensively with the REC1 and REC2 domains. The PAM-containing DNA duplex comprises target strand nucleotides dT0–dT8 and non-target strand nucleotides dA(8)*–dA0* and is contacted by the PI, WED, and REC1 domains. The 5’-TTN-3’ PAM is recognized in FnCas12a by a mechanism combining the shape- specific recognition of a narrowed minor groove, with base-specific recognition of the PAM bases by two invariant residues, Lys671 and Lys613. Directly downstream of the PAM, the duplex of the target DNA is disrupted by the side chain of residue Lys667, which is inserted between the DNA strands and forms a cation-π stacking interaction with the dA0–dT0* base pair. The phosphate group linking target strand residues dT(-1) and dT0 is coordinated by hydrogen-bonding interactions with the side chain of Lys823 and the backbone amide of Gly826. Target strand residue dT(-1) bends away from residue T0, allowing the target strand to interact with the seed sequence of the crRNA. The non-target strand nucleotides dT1*–dT5* interact with the Arg692- Ser702 loop in FnCas12a through hydrogen-bonding and ionic interactions between backbone phosphate groups and side chains of Arg692, Asn700, Ser702, and Gln704, as well as main-chain amide groups of Lys699, Asn700, and Ser702. Alanine substitution of Q704 or replacement of residues Thr698–Ser702 in FnCas12a with the sequence Ala-Gly3 (SEQ ID NO: 115) substantially reduced DNA cleavage activity, suggesting that these residues contribute to R-loop formation by stabilizing the displaced conformation of the nontarget DNA strand. [0270] In the FnCas12a R-loop complex, the crRNA-target strand heteroduplex is terminated by a stacking interaction with a conserved aromatic residue (Tyr410). This prevents base pairing between the crRNA and the target strand beyond nucleotides U20 and dA(-20), respectively. Beyond this point, the target DNA strand nucleotides re-engage the non-target DNA strand, forming a PAM-distal DNA duplex comprising nucleotides dC(-21)–dA(-27) and dG21*–dT27*, respectively. The duplex is confined between the REC2 and Nuc domains at the end of the central channel formed by the REC and NUC lobes. [0271] Target DNA cleavage: FnCpf1 can independently accommodate both the target and non-target DNA strands in the catalytic pocket of the RuvC domain. The RuvC active site contains three catalytic residues (D917, E1006, and D1255). Structural observations suggest that both the target and non-target DNA strands are cleaved by the same catalytic mechanism in a single active site in Cpf1/Cas12a enzymes. [0272] Another type V CRISPR is AsCpf1 from Acidaminococcus sp BV3L6 (Yamano et al., Crystal structure of Cpf1 in complex with guide RNA and target DNA, Cell 165, 949-962, May 5, 2016) [0273] In certain embodiments, the nuclease comprises a Cas12f effector. Small CRISPR- associated effector proteins belonging to the type V-F subtype have been identified through the mining of sequence databases and members classified into Cas12f1 (Cas14a and type V-U3), Cas12f2 (Cas14b) and Cas12f3 (Cas14c, type V-U2 and U4). (See, e.g., Karvelis et al., PAM recognition by miniature CRISPR–Cas12f nucleases triggers programmable double-stranded DNA target cleavage. Nucleic Acids Research, 21 May 2020, 48(9), 5016–23 doi.org/10.1093/nar/gkaa208). Xu et al. described development of a 529 amino acid Cas12f-based system for mammalian genome engineering through multiple rounds of iterative protein engineering and screening. (Xu, X. et al., Engineered Miniature CRISPR-Cas System for Mammalian Genome Regulation and Editing. Molecular Cell, October 21, 2021, 81(20): 4333-45, doi.org/10.1016/j.molcel.2021.08.008). [0274] Exemplary CRISPR-Cas proteins and enzymes used in the Prime Editors herein include the following without limitation. 6.3.1. Table 5. Cas12a orthologs
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
6.3.2. Table 6. Cas12b (C2c1) Orthologs
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Table 6 - Cas12b (C2c1) orthologs
Figure imgf000127_0001
6.3.3. Table 7. Cas12c (C2c3) Orthologs
Figure imgf000127_0002
Figure imgf000128_0001
Figure imgf000129_0001
6.3.4. Table 8. Cas12e (CasY) Otrhologs
Figure imgf000129_0002
Figure imgf000130_0001
6.4. Protospacer Adjacent Motif [0275] As used herein, the term “protospacer adjacent sequence” or “protospacer adjacent motif” or “PAM” refers to an approximately 2-6 base pair DNA sequence (or a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-long nucleotide sequence) that is an important targeting component of a Cas9 nuclease. Typically, the PAM sequence is on either strand, and is downstream in the 5' to 3' direction of Cas9 cut site. The canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5'-NGG-3' wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases. Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence. [0276] For example, with reference to the canonical SpCas9 amino acid sequence, the PAM specificity can be modified by introducing one or more mutations, including (a) D1135V, R1335Q, and T1337R “the VQR variant”, which alters the PAM specificity to NGAN or NGNG, (b) D1135E, R1335Q, and T1337R “the EQR variant”, which alters the PAM specificity to NGAG, and (c) D1135V, G1218R, R1335E, and T1337R “the VRER variant”, which alters the PAM specificity to NGCG. In addition, the D1135E variant of canonical SpCas9 still recognizes NGG, but it is more selective compared to the wild type SpCas9 protein. [0277] It will also be appreciated that Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) can have varying PAM specificities and in some embodiments are therefore chosen based on the desired PAM recognition. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. In addition, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW. In still another example, Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These examples are not meant to be limiting. It will be further appreciated that non- SpCas9s bind a variety of PAM sequences, which makes them useful to expand the range of sequences that can be targeted according to the invention. Furthermore, non-SpCas9s may have other characteristics that make them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged into adeno-associated virus (AAV). Further reference may be made to Shah et al., “Protospacer recognition motifs: mixed identities and functional diversity,” RNA Biology, 10(5): 891-899 (which is incorporated herein by reference). Gasiunas used cell-free biochemical screens to identify protospacer adjacent motif (PAM) and guide RNA requirements of 79 Cas9 proteins. (Gasiunas et al., A catalogue of biochemically diverse CRISPR-Cas9 orthologs, Nature Communications 11:5512 doi.org/10.1038/s41467-020-19344-1) The authors described 7 classes of gRNA and 50 different PAM requirement. [0278] Oh, Y. et al. describe linking reverse transcriptase to a Francisella novicida Cas9 [FnCas9(H969A)] nickase module. (Oh, Y. et al., Expansion of the prime editing modality with Cas9 from Francisella novicida, bioRxiv 2021.05.25.445577; doi.org/10.1101/2021.05.25.445577). By increasing the distance to the PAM, the FnCas9(H969A) nickase module expands the region of a reverse transcription template (RTT) following the primer binding site. 6.5. Gene Editor Polypeptides [0279] “Prime editor fusion protein” describes a protein that is used in prime editing. Prime editing uses CRISPR enzyme that nicks or cuts only single strand of double stranded DNA, i.e., a nickase; and a nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts. Such an enzyme can be a catalytically-impaired Cas9 endonuclease (a nickase). Such an enzyme can be a Casl2a/b, MAD7, or variant thereof. The nickase is fused to an engineered reverse transcriptase (RT). The nickase is programmed (directed) with a attachment site-containing guide RNA. The skilled person in the art would appreciate that the atgRNA both specifies the target site and encodes the desired edit. Advantageously the nickase is a catalytically-impaired Cas9 endonuclease, a Cas9 nickase, that is fused to the reverse transcriptase. During genetic editing, the Cas9 nickase part of the protein is guided to the DNA target site by the pegRNA, whereby a nick or single stranded cut occurs. The reverse transcriptase domain then uses the pegRNA to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Afterward, optionally, the prime editor (PE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA). [0280] As used herein, “PE1” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a wild type MMLV RT having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]- [linker]-[MMLV_RT(wt)] + a desired atgRNA. In various embodiments, the prime editors disclosed herein is comprised of PE1. [0281] As used herein, “PE2” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]- [linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] + a desired PEgRNA. In various embodiments, the prime editors disclosed herein is comprised of PE2. [0282] In various embodiments, the prime editors disclosed herein is comprised of PE2 and co-expression of MMR protein MLH1dn, that is PE4. [0283] As used herein, “PE3” refers to PE2 plus a second-strand nicking guide RNA that complexes with the PE2 and introduces a nick in the non-edited DNA strand. The induction of the second nick increases the chances of the unedited strand, rather than the edited strand, to be repaired. In various embodiments, the prime editors disclosed herein is comprised of PE3. [0284] In various embodiments, the prime editors disclosed herein is comprised of PE3 and co-expression of MMR protein MLH1dn, that is PE5. [0285] As used herein, “PE3b” refers to PE3 but wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence with mismatches to the unedited original allele that matches only the edited strand. Using this strategy, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place. 6.6. Guides for Prime Editing [0286] Anzalone et al., 2019 (Nature 576:149) describes prime editing and a prime editing complex using a type II CRISPR and can be used herein. A prime editing complex consists of a type II CRISPR PE protein containing an RNA-guided DNA-nicking domain fused to a reverse transcriptase (RT) domain and complexed with a pegRNA. The pegRNA comprises (5’ to 3’) a spacer that is complementary to the target sequence of a genomic DNA, a nickase (e.g. Cas9) binding site, a reverse transcriptase template including editing positions, and primer binding site (PBS). The PE–pegRNA complex binds the target DNA and the CRISPR protein nicks the PAM- containing strand. The resulting 3′ end of the nicked target hybridizes to the primer-binding site (PBS) of the pegRNA, then primes reverse transcription of new DNA containing the desired edit using the RT template of the pegRNA. The overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template/primer binding site appended to the 3’ end. The structure leaves the PBS at the 3’ end of the pegRNA free to bind to the nicked strand complementary to the target which forms the primer for reverse transcription. [0287] Guide RNAs of CRISPRs differ in overall structure. For example, while the spacer of a type II gRNA is located at the 5’ end, the spacer of a type V gRNA is located towards the 3’ end, with the CRISPR protein (e.g. Cas12a) binding region located toward the 5’ end. Accordingly, the regions of a type V pegRNA are rearranged compared to a type II pegRNA. The overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template/primer binding site appended to the 3’ end. The pegRNA comprises (5’ to 3’) a CRISPR protein-binding region, a spacer which is complementary to the target sequence of a genomic DNA, a reverse transcriptase template including editing positions, and primer binding site (PBS). [0288] In typical embodiments, the guide RNA or guide RNA complex is capable of binding a DNA binding nickase selected from the group consisting of: Cas9-D10A, Cas9-H840A, Cas12a/b/c/d/e nickase, CasX nickase, SaCas9 nickase, and CasY nickase. In certain embodiments, the nickase is linked or fused to one or more of a reverse transcriptase. In certain embodiments, the nickase is linked or fused to one or more of a reverse transcriptase and integrase. In certain embodiments, the nickase is linked or fused to one or more of an integrase. 6.7. Attachment Site-Containing Guide RNA (atgRNA) [0289] As used herein, the term "attachment site-containing guide RNA" (atgRNA) and the like refer to an extended single guide RNA (sgRNA) comprising a primer binding site (PBS), a reverse transcriptase (RT) template sequence, and wherein the RT template encodes for an integration recognition site or a recombinase recognition site that can be recognized by a recombinase, integrase, or transposase. In some embodiments, the RT template comprises a clamp sequence and an integration recognition site. As referred to herein an atgRNA may be referred to as a guide RNA. An integration recognition site or recombinase target recognition site incorporated into the pegRNA is referred to as an attachment site containing guide RNA (atgRNA). [0290] As used herein, the term “cognate integration recognition site” or “integration cognate” or “cognate pair” refers to a first integration recognition site (e.g., any of the integration recognition sites described herein) and a second integration recognition site (e.g., any of the integration recognition sites described herein) that can be recombined. Recombination between a first integration recognition site (e.g., any of the integration recognition sites described herein) and a second recognition site (e.g., any of the integration recognition sites described herein) is mediated by functional symmetry between the two integration recognition sites and the central dinucleotide of each of the two integration recognition sites. In some cases, a first integration recognition site (e.g., any of the integration recognition sites described herein) that can be recombined with a second integration recognition site (e.g., any of the integration recognition sites described herein) are referred to as a “cognate pair.” A non-limiting example of a cognate pair include an attB site and an attP site, whereby a serine integrase mediates recombination between the attB site and the attP site. FIGs.1A-1E show optimization of the integration recognition site. [0291] In typical embodiments, an atgRNA comprises a reverse transcriptase template that encodes, partially or in its entirety, an integration recognition site (also referred to as an integration target recognition site) or a recombinase recognition site (also referred to as a recombinase target recognition site). The integration target recognition site, which is to be placed at a desired location in the genome or intracellular nucleic acid, is referred to as a “beacon,” a “beacon” site or an “attachment site” or an “integration recognition site” or a “landing pad” or “landing site.” An integration target recognition site or recombinase target recognition site incorporated into the pegRNA is referred to as an attachment site containing guide RNA (atgRNA). [0292] In typical embodiments, an atgRNA comprises a reverse transcriptase template that encodes, partially or in entirety, for an integration target recognition site or recombinase target recognition site. The integration target recognition site, which is to be place at a desired location in the genome, is referred to as a “beacon site” or an “attachment site” or a “landing pad” or “landing site.” [0293] During genome editing, the primer binding site allows the 3' end of the nicked DNA strand to hybridize to the atgRNA, while the RT template serves as a template for the synthesis of edited genetic information. The atgRNA is capable for instance, without limitation, of (i) identifying the target nucleotide sequence to be edited and (ii) encoding new genetic information that replaces (or in some cases adds) the targeted sequence. In some embodiments, the atgRNA is capable of (i) identifying the target nucleotide sequence to be edited and (ii) encoding an integration site that replaces (or inserts/deletes within) the targeted sequences. [0294] In some embodiments, where the system contains a first atgRNA and a second atgRNA (see FIG.5), the first atgRNA and the second atgRNA are an at least first pair of atgRNAs, where the at least first pair of atgRNAs have domains that are capable of guiding the gene editor polypeptide or prime editor fusion protein to a target sequence, the first atgRNA further includes a first RT template that comprises at least a portion of the first integration recognition site; and the second atgRNA further includes a second RT template that comprises at least a portion of the first integration recognition site, and the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site. [0295] In some embodiments, the first atgRNA’s reverse transcriptase template encodes for a first single-stranded DNA sequence (i.e., a first DNA flap) that contains a complementary region to a second single-stranded DNA sequence (i.e., a second DNA flap) encoded by a second atgRNA comprising a second reverse transcriptase template. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 5 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 10 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 20 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 30 consecutive bases of an integrase target recognition site. Use of two guide RNAs that are (or encode DNA that is) partially complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site-containing guide RNAs (atgRNAs). In certain embodiments, use of two guide RNAs that are (or encode DNA that is) full complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site- containing guide RNAs (atgRNAs). [0296] As shown in FIGs. 1A-1E, AttP variants (SEQ ID NOS: 394 and 540-542, respectively, in order of appearance) can be optimized. [0297] Table 9 includes atgRNAs, sgRNAs and nicking guides that can be used herein. Spacers are labeled in capital font (SPACER), RT regions in bold capital (RT REGION), AttB sites in bold lower case (attb site), and PBS in capital italics (PBS). Unless otherwise denoted, the AttB is for Bxb1. 6.7.1. Table 9. atgRNAs
Figure imgf000136_0001
Table 9
Figure imgf000137_0001
Table 9
Figure imgf000138_0001
Table 9
Figure imgf000139_0001
Table 9
Figure imgf000140_0001
Table 9
Figure imgf000141_0001
Table 9
Figure imgf000142_0001
Table 9
Figure imgf000143_0001
Table 9
Figure imgf000144_0001
Table 9
Figure imgf000145_0001
Table 9
Figure imgf000146_0001
Table 9
Figure imgf000147_0001
Table 9
Figure imgf000148_0001
Table 9
Figure imgf000149_0001
Table 9
Figure imgf000150_0001
Table 9
Figure imgf000151_0001
Table 9
Figure imgf000152_0001
Table 9
Figure imgf000153_0001
Table 9
Figure imgf000154_0001
Table 9
Figure imgf000155_0001
Table 9
Figure imgf000156_0001
6.8. Integrases/Recombinases and Integration/Recombination Sites [0298] In typical embodiments, a gene editor polypeptide described herein contains an integrase or recombinase. In some embodiments, the integrase is delivered as a protein or the integrase is encoded in a delivered polynucleotide. In some embodiments, the integration enzyme is selected from the group consisting of Dre, Vika, Bxb1, ϕC31, RDF, ϕBTl, R1, R2, R3, R4, R5, TP901-1, A118, ϕFCl, ϕC1, MR11, TG1, ϕ370.l, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, ϕRV, retrotransposases encoded by a Tc1/mariner family member including but not limited to retrotransposases encoded by LI, Tol2, Tel, Tc3, Himar 1 (isolated from the horn fly, Haematobia irritans), Mos1 (Mosaic element of Drosophila mauritiana), and Minos, and any mutants thereof. As can be used herein, Xu et al describes methods for evaluating integrase activity in E. coli and mammalian cells and confirmed at least R4, φC31, φBT1, Bxb1, SPBc, TP901-1 and Wβ integrases to be active on substrates integrated into the genome of HT1080 cells (Xu et al., 2013, Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol.2013 Oct 20;13:87. doi: 10.1186/1472-6750-13-87). Durrant describes new large serine recombinases (LSRs) divided into three classes distinguished from one another by efficiency and specificity, including landing pad LSRs which outperform wild-type Bxb1 in episomal and chromosomal integration efficiency, LSRs that achieve both efficient and site- specific integration without a landing pad, and multi-targeting LSRs with minimal site-specificity. Additionally, embodiments can include any serine recombinase such as BceINT, SSCINT, SACINT, and INT10 (see Ionnidi et al., 2021; Drag-and-drop genome insertion without DNA cleavage with CRISPR directed integrases. bioRxiv 2021.11.01.466786, doi.org/10.1101/2021.11.01.466786). In some embodiments, the integration site can be selected from an attB site, an attP site, an attL site, an attR site, a lox71 site a Vox site, or a FRT site. [0299] It will be appreciated that desired activity of integrases, transposases and the like can depend on nuclear localization. In certain embodiments, prokaryotic enzymes are adapted to modulate nuclear localization. In certain embodiments, eukaryotic or vertebrate enzymes are adapted to modulate nuclear localization. In certain embodiments, the disclosure provides fusion or hybrid proteins. Such modulation can comprise addition or removal of one or more nuclear localization signal (NLS) and/or addition or removal of one or more nuclear export signal (NES). Xu et al compared derivatives of fourteen serine integrases that either possess or lack a nuclear localization signal (NLS) to conclude that certain integrases benefit from addition of an NLS whereas others are transported efficiently without addition, and a major determinant of activity in yeast and vertebrate cells is avoidance of toxicity. (Xu et al., 2016, Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiae. BMC Biotechnol. 2016 Feb 9; 16:13. doi: 10.1186/s12896-016-0241-5). Ramakrishnan et al. systematically studied the effect of different NES mutants developed from mariner-like elements (MLEs) on transposase localization and activity and concluded that nuclear export provides a means of controlling transposition activity and maintaining genome integrity. (Ramakrishnan et al. Nuclear export signal (NES) of transposases affects the transposition activity of mariner-like elements Ppmar1 and Ppmar2 of moso bamboo. Mob DNA. 2019 Aug 19;10:35. doi:10.1186/s13100-019-0179-y). The methods and constructs are used to modulate nuclear localization of system components of the invention. In typical embodiments, the integrase used herein is selected from Table 10.
Figure imgf000158_0001
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Figure imgf000165_0001
[0300] Sequences of insertion sites (i.e., recognition target sites) suitable for use in embodiments of the disclosure are presented below.
Figure imgf000165_0002
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Figure imgf000170_0001
[0301] In certain embodiments, the one or more integration recognition sites is specific for a serine integrase other than BxB1. [0302] In some embodiments, the integration enzyme is selected from one of the about 27,000 Serine integrases described in International Patent Publication No. WO 2023/070031A2, which is hereby incorporated by reference in its entirety. 6.9. Split Gene Editor Guide RNA Compositions [0303] The present disclosure contemplates a split guide RNA comprised of two or more polynucleotides that are capable of forming a guide RNA complex (see Liu et al; doi: 10.1038/s41587-022-01255-9 the entirety of which is incorporated by reference herein). In some embodiments, the guide RNA may be a guide RNA complex, wherein the guide RNA complex is comprised of at least two polynucleotide components. In certain embodiments, the guide RNA complex is comprised of a first polynucleotide component and a second polynucleotide component. In certain embodiments, the first polynucleotide component comprises a spacer complementary to a target first genomic site and a scaffold capable of binding to a DNA binding nickase, and wherein the second polynucleotide component comprises a reverse transcriptase template comprised of at least an integrase target recognition site, a primer binding site and a RNA- protein recruitment domain. In some embodiments, the RNA-protein recruitment domain is a MS2 hairpin. [0304] In some embodiments, at least one of the first polynucleotide component and the second polynucleotide component are circularized. In some embodiments, circularization is mediated by a ribozyme or ligase. In some embodiments, circularization is mediated by covalently or non-covalently linking the 5’ and 3’ termini. [0305] In certain embodiments, the guide RNA or the guide RNA complex further comprises one or more of an RNA-protein recruitment domain, RNA-RNA recruitment domain, a transcriptional termination signal, a reverse transcription termination signal, an RNA ribozyme, or a chemical linker. [0306] In typical embodiments, one or more guide RNA complex is comprised in one or more RNA polynucleotides or DNA polynucleotides. 6.10. Guide RNA Compositions for Dual Guide RNA Systems [0307] In some embodiments, the guide RNA or the guide RNA complex reverse transcriptase template encodes for a first single-stranded DNA sequence (i.e., a first DNA flap) that contains a complementary region to a second single-stranded DNA sequence (i.e., a second DNA flap) encoded by a second guide RNA comprised of a reverse transcriptase template. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 5 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 10 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single- stranded DNA sequences is comprised of more than 20 consecutive bases of an integrase target recognition site. In certain embodiments, the complementary region between the first and second single-stranded DNA sequences is comprised of more than 30 consecutive bases of an integrase target recognition site. Use of two guide RNAs that are (or encode DNA that is) partially complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site- containing guide RNAs (atgRNAs). In certain embodiments, use of two guide RNAs that are (or encode DNA that is) full complementarity to each other and comprised of consecutive bases of an integrase target recognition site are referred to as dual, paired, annealing, complementary, or twin attachment site-containing guide RNAs (atgRNAs). [0308] In some embodiments, the integrase target recognition site is an Bxb1 attB or attP sequence. In some embodiments, the att-site central dinucleotide is not GT or CA. [0309] In typical embodiments, one or more guide RNA or the guide RNA complex is comprised in one or more RNA polynucleotides or DNA polynucleotides. [0310] In typical embodiments, the one or more guide RNA complex is a split guide RNA complex or split guide RNA system. In typical embodiments, the one or more guide RNA complex is a split atgRNA complex or split atgRNA system. [0311] A non-limiting example of a dual gRNA (i.e., dual atgRNA system) is as described in FIG.5. 6.11. Programmable Gene Insertion within a Pleiopluripotent Cell Genome [0312] Described herein is a method of editing a pleiopluripotent cell genome. In typical embodiments, the pleiopluripotent cells are edited using, at least in part, the information provided in FIGs.1-5. In some embodiments, editing the pleiopluripotent cell genome results in a cell that, following differentiation and transplantation into a subject, is capable of evading a subject’s immune system (e.g., encoding for one or more exogenous polypeptides that modulate immune response) but safeguarded from runaway expansion (e.g., inducible suicide genes) (see, e.g., FIG. 2). In some embodiments, the methods described herein are designed so that beacons (and ultimately the donor polynucleotide templates) are targeted to genomic loci with the aim of reducing expression (e.g., knocking out) of the gene associated with the particular genomic locus being targeted while also serving as a genomic locus suitable for expression of the aforementioned transgenes (e.g., exogenous polypeptides capable of modulating an immune response, inducible suicide genes, or a combination thereof). The pleiopluripotent cell genome can be further modified to include additional integration recognition sites (i.e., landing pads and beacons) incorporated into the genome for integrating additional polynucleotides encoding therapeutic agents. Example 10 provides a non-limiting exemplary method for generating pleiopluripotent stem cells having a beacon (integration recognition site) in each of the B2M, CIITA, TRAC, and AAVS1 loci. The pleiopluripotent stem cells can then be used for programmable gene insertion. In some cases, this achieved using multiplex programmable gene insertion, for example, where a different donor polynucleotide template is inserted into each beacon site-specifically placed into the aforementioned loci (see, e.g., Example 11). The resulting genetically modified pleiopluripotent cells can be differentiated to a non-pleiopluripotent cell (e.g., a hematopoietic cell, a neuronal cell, a cardiac cell, or a pancreatic cell) and used as a source of material for allogeneic cell therapy. [0313] In a typical embodiment, a method of generating pleiopluripotent cells includes site- specifically incorporating at least a first integration recognition site into the genome of a pleiopluripotent cell. [0314] In some embodiments, site-specifically incorporating the at least first integration recognition site is effected by introducing into the pleiopluripotent stem cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein or a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the pleiopluripotent stem cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site- specifically incorporated into the genome of the pleiopluripotent stem cell. [0315] In some embodiments, the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is a first atgRNA that further includes a first RT template that comprises at least a portion of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a second atgRNA that further includes a second RT template that comprises at least a portion of the first integration recognition site, wherein the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site. [0316] In some embodiments, the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is an atgRNA that further includes an RT template that comprises at least a portion of the first integration recognition site, wherein the atgRNA encodes the entirety of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a nicking gRNA. [0317] In some embodiments, the methods include incorporating a plurality of integration recognition sites into the genome of a pleiopluripotent cell. In some embodiments, the method includes incorporating two integration recognition sites, three integration recognition sites, four integration recognition sites, five integration recognition sites, six integration recognition sites, seven integration recognition sites, eight integration recognition sites, nine integration recognition sites, or ten or more integration recognition sites into the genome of a pleiopluripotent cell. In some embodiments, each of the additional integration recognition sites (e.g., second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth or more) are an orthogonal pair with an integration recognition site in one or more additional donor polynucleotides. [0318] In another embodiment, a method of generating pleiopluripotent cells includes (a) site- specifically incorporating at least a first integration recognition site into the genome of a pleiopluripotent cell, wherein site-specifically incorporating the at least first integration recognition site is effected by introducing into the pleiopluripotent stem cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein or a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the pleiopluripotent stem cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site- specifically incorporated into the genome of the pleiopluripotent stem cell; and (b) integrating at least a first donor polynucleotide template into the pleiopluripotent cell genome at the first incorporated integration recognition site, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the at least one incorporated integration recognition site by the integrase; thereby producing a second generation pleiopluripotent cell. In some embodiments, incorporation of the integration recognition site into the pleiopluripotent cell genome is performed concurrently with integration of the donor polynucleotide template into the pleiopluripotent cell genome. [0319] In some embodiments, an at least first integration recognition site is incorporated into the genome of the pleiopluripotent cell using a homology directed repair, homology-independent target insertion (HITI), retrotransposon-mediated integration, viral vector-mediated integration (e.g., lentivirus), transposon-mediated integration (e.g., sleeping beauty), and/or recombinase- mediated integration (e.g., serine recombinase fC31 integrase). In such embodiments, at least a first donor polynucleotide template can be integrated into the genome of the pleiopluripotent cell at the at least first incorporated recognition site using an integrase. [0320] In another embodiment, a method of generating a pleiopluripotent cell (e.g., a second generation pleiopluripotent cell) includes integrating, into the genome of the pleiopluripotent cell at an at least first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a second generation pleiopluripotent cell. [0321] In another embodiment, a method of generating a pleiopluripotent cell (e.g., a third generation pleiopluripotent cell) includes integrating, into the genome of the pleiopluripotent cell at an at least first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the at least one incorporated genomic integration recognition sites by the integrase; and (b) site- specifically incorporating a second integration recognition site into the genome of the pleiopluripotent cell, thereby producing a third generation pleiopluripotent cell. [0322] In another embodiment, a method of generating a pleiopluripotent cell (e.g., a fourth generation pleiopluripotent cell) includes integrating, into the genome of the pleiopluripotent cell at an at least the first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the at least one incorporated genomic integration recognition sites by the integrase; (b) site- specifically incorporating a second integration recognition site into the genome of the pleiopluripotent cell; and (c) integrating a second donor polynucleotide template into the pleiopluripotent cell genome at the second incorporated integration recognition site, by delivering into the cell: (i) the second donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration recognition sites orthogonal to the second integration recognition site, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the second incorporated genomic integration recognition sites by the integrase; thereby producing a fourth generation pleiopluripotent cell. In some embodiments, incorporation of the second integration recognition site into the pleiopluripotent cell genome is performed concurrently with integration of the second donor polynucleotide template into the pleiopluripotent cell genome. [0323] In some embodiments, an integration recognition site (i.e., beacon) is incorporated into a pleiopluripotent cell genome by delivering into the cell: (i) a gene editor protein (or a polynucleotide encoding a gene editor protein) or a prime editor fusion protein (or a polynucleotide encoding a prime editor fusion protein), wherein the gene editor protein or prime editor fusion protein is capable of incorporating the integration recognition site into the genome, and (ii) two guide RNAs (e.g., two atgRNAs), wherein each of the two guide RNA encodes all or part of an integrase target recognition site. Further, the method includes integrating a donor polynucleotide template into the pleiopluripotent cell genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target site, and an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the incorporated integration recognition site by the integrase, thereby producing a genetically modified pleiopluripotent cell (e.g., a second generation pleiopluripotent cell). In some embodiments, incorporation of the integration recognition site (i.e., beacon) into the pleiopluripotent cell genome is performed concurrently with integration of the donor polynucleotide template into the pleiopluripotent cell genome. [0324] In some embodiments, an integration recognition site (i.e., beacon) is incorporated into a pleiopluripotent cell genome by delivering into the cell: (i) a gene editor protein (or a polynucleotide encoding a gene editor protein) or a prime editor fusion protein (or a polynucleotide encoding a prime editor fusion protein), wherein the gene editor protein or prime editor fusion protein is capable of incorporating the integration recognition site into the genome, and (ii) two guide RNA complexes (e.g., two complexes each comprising an atgRNA), wherein each of the two guide RNA complexes is comprised of at least one polynucleotide that encodes all or part of an integrase target recognition site. Further, the method includes integrating a donor polynucleotide template into the pleiopluripotent cell genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target site, and an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the incorporated integration recognition site by the integrase, thereby producing a genetically modified pleiopluripotent cell (e.g., a second generation pleiopluripotent cell). In some embodiments, incorporation of the integration recognition site (i.e., beacon/beacon placement) into the pleiopluripotent cell genome is performed concurrently with integration of the donor polynucleotide template into the pleiopluripotent cell genome. [0325] In some embodiments, an integration recognition site (i.e., beacon/beacon placement) is incorporated into a pleiopluripotent cell genome by delivering into the cell: (i) a gene editor protein (or a polynucleotide encoding a gene editor protein) or a prime editor fusion protein (or a polynucleotide encoding a prime editor fusion protein), wherein the gene editor protein or prime editor fusion protein is capable of incorporating the integration recognition site into the genome, (ii) one or more guide RNA, wherein the one or more guide RNA encodes an integrase target recognition site (i.e., atgRNA), and (iii) optionally, a nicking guide RNA (ngRNA). Further, integrating a donor polynucleotide template into the pleiopluripotent cell genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target site, and an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the incorporated integration recognition site by the integrase, thereby producing a genetically modified pleiopluripotent cell (e.g., a second generation pleiopluripotent cell). In some embodiments, incorporation of the integration recognition site (i.e., beacon/beacon placement) into the pleiopluripotent cell genome is performed concurrently with integration of the donor polynucleotide template into the pleiopluripotent cell genome. [0326] In some embodiments, an integration recognition site (i.e., beacon/beacon placement) is incorporated into a pleiopluripotent cell genome by delivering into the cell: (i) a gene editor protein (or a polynucleotide encoding a gene editor protein) or a prime editor fusion protein (or a polynucleotide encoding a prime editor fusion protein), wherein the gene editor protein or prime editor fusion protein is capable of incorporating the integration recognition site into the genome, (ii) one or more guide RNA complex, wherein the one or more guide RNA complex is comprised of at least one polynucleotide that encodes an integrase target recognition site (i.e., atgRNA), and (iii) optionally, a nicking guide RNA (ngRNA). Further, integrating a donor polynucleotide template into the pleiopluripotent cell genome at the incorporated target recognition site by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of an integration target site, and an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the incorporated integration recognition site by the integrase, thereby producing a genetically modified pleiopluripotent cell (e.g., a second generation pleiopluripotent cell). In some embodiments, incorporation of the integration recognition site (i.e., beacon/beacon placement) into the pleiopluripotent cell genome is performed concurrently with integration of the donor polynucleotide template into the pleiopluripotent cell genome. [0327] In certain embodiments, the pleiopluripotent cell is a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, the pleiopluripotent cell is a human cell. 6.11.1. Knockout of HLA Class I and/or Class II proteins [0328] In certain embodiments, the pleiopluripotent comprises a genetic perturbation that results in reduction of an allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell. In some embodiments, at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell. In some embodiments, the pleiopluripotent cell includes a plurality of integration recognition sites at least one of which is incorporated into the pleiopluripotent cell genome at a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell. Non-limiting examples of loci, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell, are as described in U.S. Patent No. 10,968,426, which is herein incorporated by reference in its entirety. [0329] In certain embodiments, the pleiopluripotent stem cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. In such cases, the deletion or reduced expression results from knockout or knockdown using base editing, prime editing, other CRISPR/Cas9-mediated methods, TALE- based methods, Zinc Finger Nuclease-based methods, siRNA, miRNA, RNAi, or a combination thereof. In some embodiments, the pleiopluripotent stem cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof as the result of knockout or knockdown of genes expressed from one or more chromosomal regions selected from: HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA- DQ. As described herein, in some embodiments, the pleiopluripotent stem cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof, as a result of incorporation of one or more integration recognition sites into a genomic loci that when disrupted leads to deletion or reduced expression of the associated gene. [0330] In some embodiments, at least one of the at least first integration recognition sites is incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof. In such cases, the pleiopluripotent cell has reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. Additionally, incorporation of the at least first integration recognition site results in reduction of a potential allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell (when administered to a patient in need thereof). [0331] In some embodiments, at least one of the at least first integration recognition sites is incorporated into the genome at a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ. In such cases, the pleiopluripotent cell has reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. Additionally, incorporation of the at least first integration recognition site results in reduction of a potential allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell (when administered to a patient in need thereof). [0332] In some embodiments, at least one of the at least first integration recognition sites is incorporated into the genome at a locus selected from B2M locus, CIITA locus, HLA-A locus, HLA- B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus. In such cases, incorporation of the at least first integration recognition site results in reduction of a potential allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell (when administered to a patient in need thereof). [0333] In some embodiments, at least one of the at least first integration recognition sites is incorporated into the genome at the B2M locus. In such cases, incorporation of the at least first integration recognition site at the B2M locus results in reduction of a potential allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell (when administered to a patient in need thereof). [0334] In some embodiments, at least one of the at least first integration recognition sites is incorporated into the genome at the CIITA locus. In such cases, incorporation of the at least first integration recognition site at the CIITA locus results in reduction of a potential allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell (when administered to a patient in need thereof). [0335] In some embodiments, at least one of the at least first integration recognition sites is incorporated into the genome at locus associated with blood group antigens, disruption of which is capable of reducing expression of the blood group antigens by the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell. In some embodiments, at least one of the at least first integration recognition sites is incorporated into the genome at an alleles of the ABO gene (alpha 1-3-N-acetylgalactosaminyltransferase and alpha 1-3-galactosyltransferase), including, without limitation ABO*A1.01 and ABO* B.01. A non-limiting example of loci, disruption of which is capable of reducing expression of the blood group antigens by the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell, are as described in U.S. Patent No.11,162,079, which is herein incorporated by reference in its entirety. [0336] In some embodiments, the pleiopluripotent cell is an iPSC derived from a healthy donor in order to minimize immunity against ABO antigens. In some embodiments, the pleiopluripotent cell is an iPSC derived from a healthy donor with blood type O. 6.11.2. Incorporation of Integration Recognition Sites into the Genome of a Pleiopluripotent Stem Cell [0337] In some embodiments, an integration recognition site is incorporated (i.e., beacon placement) into a pleiopluripotent cell genome using a single atgRNA and a single nicking guide RNA (ngRNA) (see, e.g., FIG. 5). In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome using two atgRNAs (dual atgRNAs) (see, e.g., FIG. 5). Beacon placement efficiency can be determined, for example, by a two-color digital droplet PCR assay that compares signal from no-insertion amplicons (i.e., wild type) with signal from beacon inserted/placed amplicons. Beacon placement efficiency can be determined, for example, by amplicon-sequencing (amp-seq). [0338] In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or greater. [0339] In some embodiments, at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a locus encoding an exon. In such embodiments, the exon is coding sequence of a gene of interest, wherein incorporation of the integration recognition site into the exon is capable of reducing or deleting expression of the gene of interest. In some embodiments, incorporation of integration recognition site at one or more exons associated with a gene of interest results in reduced expression of at least 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the gene of interest as compared to a cell where the integration recognition site is not incorporated at one or more exons associated with the gene of interest. [0340] In some embodiments, efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at an exon of a gene of interest depends on the molar ratio of a single atgRNA and a single ngRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 900 picomoles total of atgRNA plus ngRNA. In some embodiments, an integration recognition site is incorporated into a pluripotent cell genome at an exon of a gene of interest using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 100 to about 1000 picomoles total of atgRNA plus ngRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein). [0341] In some embodiments, efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at an exon of a gene of interest depends on the molar ratio of each atgRNA in a dual atgRNA method. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest using dual atgRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of each atgRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest using dual atgRNAs at a 1:1 molar ratio of each atgRNA at about 100 to about 1000picomoles total of atgRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest using dual atgRNAs at a 1:1 molar ratio of each atgRNA at about 100 to about 1000 picomoles total of atgRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein). [0342] In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using a single atgRNA and a single ngRNA. [0343] In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at an exon of a gene of interest at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using dual atgRNAs. [0344] In some embodiments, the integration recognition site is incorporated at one or more loci associated with HLA Class I proteins, one or more loci associate with HLA Class II proteins, or a combination thereof. In some embodiments, the integration recognition site is incorporated at one or more loci associated with HLA Class I proteins. In some embodiments, the integration target site is incorporated at one or more loci associated with HLA Class II proteins. [0345] In some embodiments, incorporation of integration recognition site at one or more loci associated with HLA Class I proteins results in reduced expression of the one or more HLA Class I proteins as compared to a cell where the integration recognition site is not incorporated at one or more loci associated with HLA Class I proteins. In some embodiments, incorporation of integration recognition site at one or more loci associated with HLA Class I proteins results in reduced expression of at least 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% for each of the one or more HLA Class I proteins as compared to a cell where the integration recognition site is not incorporated at one or more loci associated with HLA Class I proteins. [0346] In some embodiments, incorporation of integration target recognition at one or more loci associated with HLA Class II proteins results in reduced expression of the one or more HLA Class II proteins as compared to a cell where the integration recognition site is not incorporated at one or more loci associated with HLA Class II proteins. In some embodiments, incorporation of integration recognition site at one or more loci associated with HLA Class II proteins results in reduced expression of at least 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% for each of the one or more HLA Class II proteins as compared to a cell where the integration recognition site is not incorporated at one or more loci associated with HLA Class II proteins. [0347] In certain embodiments, the integration recognition site is incorporated at one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. In some embodiments, the integration recognition target site is incorporated at one or more of the B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus. In some embodiments, a donor polynucleotide is integrated at one or more incorporated integration target site. [0348] In some embodiments, efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at the B2M locus depends on the molar ratio of a single atgRNA and a single ngRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 900 picomoles total of atgRNA plus ngRNA. In some embodiments, an integration recognition site is incorporated into a pluripotent cell genome at the B2M locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 100 to about 1000 picomoles total of atgRNA plus ngRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein). [0349] In some embodiments, efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at the B2M locus depends on the molar ratio of each atgRNA in a dual atgRNA method. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus using dual atgRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of each atgRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus using dual atgRNAs at a 1:1 molar ratio of each atgRNA at about 100 to about 1000picomoles total of atgRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus using dual atgRNAs at a 1:1 molar ratio of each atgRNA at about 100 to about 1000 picomoles total of atgRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein). [0350] In some embodiments, efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at the CIITA locus depends on the molar ratio of a single atgRNA and a single ngRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 900 picomoles total of atgRNA plus ngRNA. In some embodiments, an integration recognition site is incorporated into a pluripotent cell genome at the CIITA locus using a single atgRNA and a single ngRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of atgRNA to ngRNA at about 100 to about 1000 picomoles total of atgRNA plus ngRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein). [0351] In some embodiments, efficiency of incorporating an integration recognition site into a pleiopluripotent cell genome at the CIITA locus depends on the molar ratio of each atgRNA in a dual atgRNA method. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus using dual atgRNA at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of each atgRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus using dual atgRNAs at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of each atgRNA at about 100 to about 1000 picomoles total of atgRNA. In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus using dual atgRNAs at a 1:1, 1.5:1, 2:1, 3:1, 1:3: 1:2, 1:1.5 or 1:1 molar ratio of each atgRNA at about 100 to about 1000 picomoles total of atgRNA and about 1 to about 10 micrograms of mRNA encoding a prime editor protein (or a primer editor fusion protein). [0352] In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using a single atgRNA and a single ngRNA. [0353] In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the CIITA locus at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using dual atgRNAs. [0354] In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using a single atgRNA and a single ngRNA. [0355] In some embodiments, an integration recognition site is incorporated into a pleiopluripotent cell genome at the B2M locus at an efficiency of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% using dual atgRNAs. [0356] In certain embodiments, an integration recognition site is specific for an integrase. In some embodiments, the integration recognition site is specific for a recombinase or transposase. In typical embodiments, the integration recognition site is an attB or attP site. In some embodiments, the integration recognition site is a modified or orthogonal attB or attP site. In certain embodiments, the integrase target recognition site is a Bxb1 attB or attP sequence. In some embodiments, the att-site central dinucleotide is not GT or CA. [0357] In some embodiments, the integration recognition site is comprised of 38 or 46 nucleotides. In some embodiments, the integration recognition site is less than 46 nucleotides. In some embodiments, the integration recognition site is less than 40 nucleotides. In some embodiments, the integration recognition site is less than 35 nucleotides. In some embodiments, the integration recognition site is less than 30 nucleotides. In some embodiments, the integration recognition site is less than 25 nucleotides. [0358] In some embodiments, the one or more guide RNA or the one or more guide RNA complex comprises a chemical modification. In certain embodiments, the chemical modification is selected from one or more of a 2’ O-methyl and phosphorothioate. [0359] Sequences of guide RNAs (i.e., attachment site-containing guide RNAs) suitable for partial replacement use in embodiments of the disclosure are presented below. In the below table: [0360] As described herein non-modified RNA are entered as ‘r_’. For example, as rA or rU. As described herein, 2’ O-methyl RNA are entered as ‘m_’. As described herein, phosphorothioated RNA are entered as ‘r_*’. As described herein, phosphorothioated 2’ O-methyl RNA are entered as ‘m_*’ [0361] Table 12 includes dual atgRNAs for incorporating an integration recognition site at the B2M locus or CIITA locus. Table 12
Figure imgf000187_0001
Table 12
Figure imgf000188_0001
Table 12
Figure imgf000189_0001
[0362] In typical embodiments, the guide RNA (e.g., atgRNA) for beacon placement comprises, in 5’ to 3’ order, (i) a spacer complementary to a first target genomic site, (ii) a scaffold capable of binding to a DNA binding nuclease or nuclease with nickase activity (or cleaves activity), (iii) a reverse transcriptase template comprised of at least an integrase target recognition site (e.g., all or a portion of an integrase target recognition site), and (iv) a primer binding site. In typical embodiments, the reverse transcriptase template encodes for a first single-stranded DNA sequence or first DNA flap sequence. 6.11.3. Donor Polynucleotide Template [0363] In certain embodiments, a cellular integrated polynucleotide (i.e., donor polynucleotide template) encodes for one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, and one or more adapters (tags), or a combination thereof. (See, e.g., FIG.3A). [0364] In some embodiments, a cellular integrated polynucleotide (i.e., donor polynucleotide template) includes one or more integration recognition sites. In some embodiments, the one or more integration recognition sites encoded in the cellular integrated polynucleotide (i.e., donor polynucleotide template) can be used to integrate a second cellular integrated polynucleotide (e.g., a second donor polynucleotide template) in the genomic loci where the first cellular integrated polynucleotide integrated. In some embodiments, the one or more integration recognition sites encoded in the cellular integrated polynucleotide (i.e., donor polynucleotide template) can be used to excise all or a portion of the cellular integrated polynucleotide integrated into pleiopluripotent cell genome. [0365] In some embodiments, expression of the one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof, is driven by a pleiopluripotent cell-specific regulatory elements (e.g., a promoter, an enhancer, or a combination thereof). Non-limiting examples of pleiopluripotent cell-specific promoters include a Nanog promoter, a Oct4 promoter, an hTERT promoter, and a Sox2 promoter. [0366] In some embodiments, expression of the one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof, is driven by a constitutive promoter. Non-limiting examples of constitutive promoters include CMV, EF1A, SV40, PGK, CAG, and UBC. [0367] In some embodiments, the first donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 1 kilobase (kb), at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, at least 30kb, at least 31kb, at least 32kb, at least 33kb, at least 34kb, or at least 35kb or more. In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 5 kilobases (kb). In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 10 kilobases (kb). In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 15 kilobases (kb). In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 20 kilobases (kb). In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 25 kilobases (kb). In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 30 kilobases (kb). In some embodiments, the first donor polynucleotide template integrated into pleiopluripotent cell genome is at least 35 kilobases (kb). [0368] In some embodiments, the first donor polynucleotide template integrated into the pleiopluripotent cell genome is at least at least 1 kilobase (kb), at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, at least 30kb, at least 31kb, at least 32kb, at least 33kb, at least 34kb, or at least 35kb or more. In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 5 kilobases (kb). In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 10 kilobases (kb). In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 15 kilobases (kb). In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 20 kilobases (kb). In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 25 kilobases (kb). In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 30 kilobases (kb). In some embodiments, the second donor polynucleotide template integrated into pleiopluripotent cell genome is at least 35 kilobases (kb). 6.11.3.1 Inducible Suicide Genes [0369] In certain embodiments, the cellular integrated polynucleotide (i.e., donor polynucleotide template) encodes for one or more inducible suicide genes. As used herein, the term “inducible suicide gene” refers to an engineered polypeptide designed to prevent the adverse events of cell therapy. In some embodiments, the inducible suicide gene is conditionally controlled to address adverse events of cell therapy (e.g., safety concerns). Conditional regulation can include control through a small molecule-mediated post-translational activation, tissue-specific regulation, temporal transcriptional regulation, or a combination thereof. The inducible suicide gene could mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation and/or antibody-mediated depletion. In some embodiments, the inducible suicide gene is activated by an exogenous molecule, e.g., a prodrug, that when activated, triggers apoptosis and/or cell death of a therapeutic cell. Non- limiting examples of inducible suicide genes include to suicide genes such as caspase 9 (or caspase 3 or 7), herpes simplex virus thymidine kinase (HSV-TK), cytosine deaminase, modified EGFR, nitroreductase, purine nucleoside phosphorylase, horseradish peroxidase, a HER1 transgene, a RQR8 transgene, or a combination thereof. In this strategy, a prodrug (e.g., an exogenous molecule) that is administered in the event of an adverse event is activated by the suicide-gene product and kills the transduced cell. [0370] In one embodiment, the one or more inducible suicide genes is caspase9. In one embodiment, the one or more inducible suicide genes comprises two caspase9 proteins where each caspase9 protein is operably linked to a different promoter sequence (See FIG. 3A). Relying on two different promoters enables both time and tissue specific control over expression of each caspase9 protein. In one embodiment, each caspase 9 protein is fused to a different controllable domain (e.g., FKBP or mFRM and FKBP), thereby generating controllable caspase9 proteins. This enables individual control over activity of each caspase9 protein (see FIG. 4). Activity of a controllable caspase9 proteins is controlled using an exogenous molecule. In one embodiment, the exogenous molecule is AP20187 (or an analog thereof). AP20187 enables homodimerization of Caspase9-FKBP fusions. A non-limiting example, as shown in FIG.4, illustrates caspase9-FKBP fusions where expression is driven by a nanog promoter. In this case, the caspase9 FKBP protein is expressed while the cells are in a pluripotent state. Contacting the cell with AP20187 during the pluripotent state results in cell death. In some embodiments, the exogenous molecule is AP21967 (or an analog thereof). A non-limiting example, as shown in FIG.4, illustrate caspase9-FKBP and caspase9-mFRB where expression is driven by a beta-actin promoter (or another constitutively active promoter). In this case, the Caspase9-FKBP and Caspase9-mFRB fusion are expressed constitutively. Therefore, contacting the cells with the AP21967 results in cell death. Either AP20187 and/or AP21967 can be administered to a subject who was previously been administered cells containing the HSV-TK gene. [0371] In one embodiment, the one or more inducible suicide genes is herpes simplex virus thymidine kinase (HSV-TK). HSV-TK converts ganciclovir into a toxic product and allows selective elimination of cells expressing the HSV-TK gene. Ganciclovir can be administered to a subject who was previously been administered cells containing the HSV-TK gene. In such cases, ganciclovir is used to eliminate the previously administered cells. [0372] In one embodiment, the donor polynucleotide templates comprise three suicide genes, including a first caspase9 protein fused to a FKBP domain operably linked to a Nanog promoter, a second caspase9 protein used to a mFRB domain and a FKBP domain operably linked to a Beta- actin promoter, and a thymidine kinase (see, e.g. FIG. 3A). In some embodiments, each of the inducible suicide genes is operably linked to a promoter. In some embodiments, the thymidine kinase is linked to one of the caspase9 proteins by a 2A self-cleaving peptide (e.g., a P2A, E2A,T2A, and F2A) or an internal ribosome entry site (IRES) (see, e.g., FIG.3A). [0373] In some embodiments, integrating a donor polynucleotide template encoding one or more inducible suicide genes into the pleiopluripotent cell genome at the one or more incorporated target recognition sites results in increased expression of the one or more inducible suicide genes as compared to a pluripotent stem genome not having a donor polynucleotide template integrated into the cell’s genome at the one or more incorporated target recognition sites. In some embodiments, integrating a donor polynucleotide template encoding one or more inducible suicide genes into the pleiopluripotent cell genome at the one or more incorporated target recognition sites results in increased expression of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more of the one or more inducible suicide genes as compared to a pluripotent stem genome not having a donor polynucleotide template integrated into the cell’s genome at the one or more incorporated target recognition sites. [0374] In some embodiments, the encoded one or more inducible suicide genes further comprises an integrase target recognition site. In some embodiments, the encoded one or more inducible suicide genes further comprises at least two integrase target recognition sites. In certain embodiments, the one or more integrase target recognition sites flank the encoded one or more inducible suicide genes. In certain embodiments, the flanking integrase target recognition sites allow for excision of the one or more inducible suicide genes. 6.11.3.2 Exogenous Polypeptides Capable of Modulating an Immune Response [0375] In certain embodiments, the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells or cells derived therefrom. In some embodiments, the one or more exogenous polypeptides capable of modulating an immune response prevent an immune response to the pleiopluripotent cells (or cells derived therefrom) by inhibiting, blocking, or preventing recognition of the pleiopluripotent cell by macrophages, T-cells, Natural Killer (NK)-cells, antigen-presenting cells, or a combination thereof. [0376] In some embodiments, the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL-1, HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof. [0377] In some embodiments, integrating a donor polynucleotide template encoding one or more exogenous polypeptides capable of modulating an immune response into the pleiopluripotent cell genome at the one or more incorporated target recognition sites results in increased expression of the one or more polypeptides capable of modulating an immune response as compared to a pluripotent stem genome not having a donor polynucleotide template integrated into the cell’s genome at the one or more incorporated target recognition sites. In some embodiments, integrating a donor polynucleotide template encoding one or more exogenous polypeptides capable of modulating an immune response into the pleiopluripotent cell genome at the one or more incorporated target recognition sites results in increased expression of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more of the one or more inducible suicide genes as compared to a pluripotent stem genome not having a donor polynucleotide template integrated into the cell’s genome at the one or more incorporated target recognition sites. [0378] In certain embodiments, the cellular integrated polynucleotide (i.e., donor polynucleotide template) encodes for two or more exogenous polypeptides capable of modulating an immune response where the sequence encoding the two or more exogenous polypeptides are separated by a 2A self-cleaving peptide (e.g., a P2A, E2A,T2A, and F2A). In other embodiments, the sequences encoding the two or more exogenous polypeptides are separated by an internal ribosome entry site (IRES). In some embodiments, a cellular integrated polynucleotide includes: CD47, PDL-1, and B2M-HLA-E. In such cases, the sequences encoding CD47, PDL-1 and B2M- HLA-E are separated by either a 2A self-cleaving peptide (e.g., a P2A, E2A,T2A, and F2A) or an IRES sequence. A non-limiting example of a cellular integrated polynucleotide is described in FIG.3A. [0379] In certain embodiments, the encoded one or more exogenous polypeptides capable of modulating an immune response further comprises an integrase target recognition site. In some embodiments, the encoded one or more exogenous polypeptides capable of modulating an immune response further comprises at least two integrase target recognition sites. In certain embodiments, the one or more integrase target recognition sites flank the encoded one or more exogenous polypeptides capable of modulating an immune response. In certain embodiments, the flanking integrase target recognition sites allow for excision of the one or more exogenous polypeptides capable of modulating an immune response. [0380] In some embodiments, the one or more exogenous polypeptides capable of modulating an immune response are capable of serving as a safety switch target. In some embodiments, the one or more exogenous polypeptides can be targeted with an antibody that is capable of specifically binding to the one or more exogenous polypeptides, thereby blocking the polypeptides ability to prevent an immune response to the pleiopluripotent cells, or cells derived therefrom. For example, a pleiopluripotent cell comprising an exogenous CD47 on it surface can be contacted with an anti- CD47 antibody, wherein the anti-CD47 antibody blocks CD47 interaction with macrophage signal regulatory protein (SIRPα), thereby allowing recognition and elimination of the cell by macrophages. In another example, the pleiopluripotent cell comprising an exogenous PD-L1 on it surface can be contacted with an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody blocks PD-L1 interaction with PD-1, thereby allowing recognition and elimination of the cell by T cells. 6.11.3.3 Adapters/Tags [0381] In some embodiments, a first or second cellular integrated polynucleotide (i.e., a first or second donor polynucleotide template) includes a sequence encoding a tag that enables isolation of the pleiopluripotent cell. The tag can be a stand-alone molecule such as a polypeptide expressed on the surface of the pleiopluripotent cell or a cell-derived from the pleiopluripotent cell. Non- limiting example of stand-alone tags includes non-natively expressed receptors (e.g., nerve truncated growth factor receptor and truncated nerve growth factor receptor). [0382] In other cases, the tag can be an amino acid sequence fused to an amino acid sequence of an endogenous protein that is expressed by a pleiopluripotent cell or a cell-derived from the pleiopluripotent cell. Non-limiting examples of tags that can be used to isolate a cell having a first or second cellular integrated polynucleotide include an ALFA tag system (e.g., SRLEEELRRRLTE (SEQ ID NO: 615)). In one non-limiting example, the ALFA tag is fused to the N-terminus of CD47 (see FIG.3A). [0383] In one embodiment, the first or second cellular integrated polynucleotide includes a sequence encoding an ALFA tag fused to one or more polypeptide expressed on the surface of the cell (e.g., one of more of the exogenous polypeptides capable of modulating an immune response). In such cases, a nanobody designed to bind specifically to the ALFA can be used to bind and isolate the pleiopluripotent cells having the tag on the surface. In some cases, an ALFA tag can be used to identify pleiopluripotent cells that include the first and/or second cellular integrated polynucleotide integrated into its genome. In some cases, an ALFA nanobody (NbALFA) is fused to one or more polypeptides on the surface of the cell, one or more ALFA tagged polypeptides can be used to engage with NbALFA fusion polypeptides for therapeutic purposes. 6.11.4. Additional Integration Recognition Sites Incorporated into Pleiopluripotent Cell Genome [0384] In some embodiments, an additional integration recognition site is incorporated into a pleiopluripotent cell genome by delivering into the cell: (i) a gene editor protein (or a polynucleotide encoding a gene editor protein) or a prime editor fusion protein (or a polynucleotide encoding a prime editor fusion protein), wherein the gene editor protein or prime editor fusion protein is capable of incorporating the integration recognition site into the genome, (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the pleiopluripotent stem cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site-specifically incorporated into the genome of the pleiopluripotent stem cell. [0385] In some embodiments, the additional integration recognition site is incorporated into the pleiopluripotent cell genome at a locus encoding an exon. In such embodiments, the exon is coding sequence of a gene of interest, wherein incorporation of the integration recognition site into the exon is capable of reducing or deleting expression of the gene of interest. [0386] In some embodiments, the additional integration recognition is incorporated into the pleiopluripotent cell genome at a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent cell. Non-limiting examples of loci, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent cell, are described herein. [0387] Incorporating an additional integration recognition site into a pleiopluripotent cell genome enables subsequent round of genetic modification of the pleiopluripotent cell genome. For example, in a subsequent round of genetic modification, a second donor polynucleotide can be integrated at the genomic locus where the additional integration recognition sites were incorporated in the early round of editing. [0388] In some embodiments, a genetically modified pleiopluripotent cell comprises one, two, three, four, five, six, seven, eight, nine, ten, or more integration recognition sites. In some embodiments, the at least one, at least two, at least three, at least four, at least five or more of the integration recognition sites can be used for placing a second donor polynucleotide template into the pleiopluripotent cell genome. 6.11.5. A Second Donor Polynucleotide Template [0389] In certain embodiments, a second donor polynucleotide template (e.g., a second cellular integrated polynucleotide) that encodes for one or more of the therapeutic agents described herein is incorporated into the pleiopluripotent cell genome. In such cases, the second cellular integrated polynucleotide (i.e., second donor polynucleotide template) can be incorporated into the pleiopluripotent cell genome at an additional integration recognition site (e.g., any of the additional integration recognition sites described herein). [0390] In some embodiments, a second donor polynucleotide template encodes transcription factors, receptors, chimeric antigen receptors, signaling molecules, secreted proteins, cytokines, enzymes, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, and persistence and/or survival of the iPSCs or cells derived from the iPSC. [0391] In some embodiments, a second donor polynucleotide template encodes for one or more of a chimeric antigen receptor, a T cell receptor, a cytokine receptor, a chemokine receptor, or a modified receptor. In some embodiments, the second donor polynucleotide template encodes for an exogenous T cell receptor. In some embodiments, the second cellular integrated polynucleotide encodes for a secreted cytokine. In some embodiments, the second donor polynucleotide template encodes for a recombinant protein. In some embodiments, the second donor polynucleotide template further comprises one or more of a co-stimulatory receptor such suitable co-stimulatory signaling regions are also well known in the art, and include members of the B7/CD28 family such as B7-1, B7-2, E7-H1, B7-H2, E7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2 or PDCD6; or ILT/CD85 family proteins such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3 or LILRB4; or tumor necrosis factor (TNF) superfamily members such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, OX40, RELT, TACI, TL1A, TNF-alpha or TNF RII; or members of the SLAM family such as 2B4, BLAME, CD2, CD2F-10, CD48, CD8, CD84, CD229, CRACC, NTB-A or SLAM; or members of the TIM family such as TIM-1, TIM-3 or TIM-4; or other co- stimulatory molecules such as CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin- 1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3 or TSLP R. [0392] The selection of the co-stimulatory signaling regions may be selected depending upon the particular use intended for the transformed cells. In particular, the co-stimulatory signaling regions selected for those which may work co-operatively or synergistically together. For example, the co-stimulatory signaling regions may be selected from CD28, CD27, ICOS, 4-1BB, OX40, CD30, GITR, HVEM, DR3 or CD40. [0393] In some embodiments, the encoded one or more chimeric antigen receptor, T cell receptor, cytokine receptor, chemokine receptor, or modified receptor, further comprises one or more integrase target recognition site. In some embodiments, the encoded one or more chimeric antigen receptor, T cell receptor, cytokine receptor, chemokine receptor, or modified receptor, further comprises at least two integrase target recognition sites. In certain embodiments, the one or more integrase target recognition sites flank the encoded one or more chimeric antigen receptor, T cell receptor, cytokine receptor, chemokine receptor, or modified receptor. In certain embodiments, the flanking integrase target recognition sites allow for excision of any inserted genetic sequence. [0394] In some embodiments, a first or second donor polynucleotide template encodes one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. In some embodiments, the first or second donor polynucleotide template encodes one or more HLA Class I proteins or one or more HLA Class II proteins that has reduced expression (e.g., due to a mutation or due to genetic ablation using a knockdown-mediated technique) in the pleiopluripotent cell or cell differentiated from the pleiopluripotent cell. In some embodiments, the HLA Class 1 protein is a B2M protein. In some embodiments, a first or second donor polynucleotide template includes an inducible promoter that controls expression of the one or more HLA Class I proteins, the one or more HLA Class II proteins, or a combination thereof. In such cases, the one or more HLA Class I proteins or one or more HLA Class II proteins can be “switched-on” at a later time point. Non-limiting examples of inducible promoter sequences that can be used in the methods described herein include a tetracycline-inducible promoter, an alcohol regulated promoter (e.g., AlcA promoter and AlcR activator), and steroid-regulated promoter (e.g., a LexA promoter and a XVE activator), a temperature-inducible promoter (e.g., Hsp70 or Hsp90), and a light-inducible promoter (e.g., FixK2 promoter and blue-light sensing protein YFI). In some embodiments, the inducible promoter is a tetracycline inducible promoter. [0395] A non-limiting example of a donor polynucleotide template that can be incorporated into the genome of a pleiopluripotent cell using the methods described herein includes the donor polynucleotide templates are as described in FIGs.3A-3B, FIG.17, and FIG.18 (see also SEQ ID NOs: 616 and 617). [0396] In some embodiments, a first or second cellular integrated polynucleotide (i.e., a first or second donor polynucleotide template) that includes an inducible promoter that controls expression of the one or more HLA Class I proteins, the one or more HLA Class II proteins, or a combination thereof, is incorporated into a pleiopluripotent cell’s genome at locus that allows for inducible gene expression can be “switched-on” at a later time point. Non-limiting criteria for genomic loci that enable inducible gene expression at a later time point include those that remain accessible to transcriptional machinery (i.e., capable of being expressed) when the cell is in a pluripotent state as well as when the cells are differentiated to one or more of the cell types described herein (e.g., a hematopoietic cell, a neuronal cell, a cardiac cell, and a beta-cell). A non- limiting example of a genomic loci that fits this criteria is the CIITA locus. 6.11.6. Safe Harbor Loci and Genomic Safe Harbor [0397] In some embodiments, an integration recognition site (i.e., beacon) is incorporated into a pleiopluripotent cell genome at a safe harbor locus or a genomic safe harbor. In such cases, the donor polynucleotide template is integrated into the pleiopluripotent cell genome at the safe harbor loci or the genomic safe harbor. [0398] In some embodiments, an additional integration recognition site (e.g., a second integration recognition site) is incorporated into a pleiopluripotent cell genome at a safe harbor locus or a genomic safe harbor. In such cases, the second donor polynucleotide template described herein) is integrated into the pleiopluripotent cell genome at the safe harbor loci or the genomic safe harbor. [0399] Safe harbor loci, safe harbor site, and genomic safe can be used interchangeably. In some embodiments, a safe harbor locus is region of the human genome (e.g., intragenic or extragenic regions) that are able to accommodate predictable expression of newly integrated DNA without adverse effects on the host cell or organism. In typical embodiments, a useful safe harbor must permit sufficient transgene expression to yield desired levels of the proteins encoded in the donor template polynucleotide. In some embodiments, a safe harbor also must not predispose cells to malignant transformation nor alter cellular functions. Non-limiting examples of criteria that describe a safe harbor loci and/or genomic safe harbor include (i) absence of disruption of regulatory elements or genes, as judged by sequence annotation; is an intergenic region in a gene dense area, or a location at the convergence between two genes transcribed in opposite directions; (ii) keep distance to minimize the possibility of long-range interactions between vector-encoded transcriptional activators and the promoters of adjacent genes, particularly cancer-related and microRNA genes; and (iii)ubiquitous transcriptional activity, as reflected by broad spatial and temporal expression patterns (e.g., as measured by expression analysis), chromatin accessibility patterns (e.g., as measured by ATAC-seq), or methylation patterns (e.g., as measured by whole- genome bisulfite sequencing), indicating ubiquitous transcriptional activity. In typical embodiments, it is important for the safe harbor loci or genomic safe harbor to have the potential for ubiquitous expression because pleiopluripotent cells during differentiation experience changes in chromatin accessibility and/or remodeling that can lead to silencing of some loci and potential activation of others. [0400] Non-limiting examples of genomic safe harbor include those as described in Aznauryan et al. (Cell Reports Methods, Discovery and validation of human genomic safe harbor sites for gene and cell therapies, 2(1): 100154 (2022); see Table S1, including Genomic coordinates: chromosome 1: 195338589-195818588 and chromosome 3: 22720711-22761389). [0401] Other non-limiting examples of safe harbor sites include adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 ( CCR5 ) gene locus, the human orthologue of the mouse ROSA26 locus, collagen and FITRP. A frequently used human SHS is the AAVS1 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion. Another locus comprises the human homolog of the murine Rosa26 locus. Yet another SHS comprises the human H11 locus on chromosome 22. In some cases, a complete gene may be prohibitively large and replacement of an entire gene impractical. 6.11.7. Therapeutic Agents [0402] This disclosure provides compositions and methods for correcting or replacing genes or gene fragments (including introns or exons) or inserting genes in new locations. In certain embodiments, such a method comprises recombination or integration of a therapeutic agent into a safe harbor site (SHS) (e.g., any of the safe harbor sites described herein). In certain embodiments, a method of the invention comprises recombining corrective gene fragments into a defective locus. [0403] The methods and compositions can be used to target, without limitation, pleiopluripotent cells, for example embryonic stem cells or induced pleiopluripotent cells (iPSCs) cells at various stages of potency. [0404] In certain embodiments, correcting or replacing genes or gene fragment (including introns and exons) includes integrating the corrective gene or fragment (the therapeutic agent) into one or more additional integration recognition sites previously incorporated into the pleiopluripotent cell genome. For example, the genes and targets described herein are encoded in the first donor template nucleotide, the second donor template nucleotide, or both. In such cases, the genes and targets described herein are integrated into the pleiopluripotent cell genome at the corresponding loci in which the integration target recognition sequences have been integrated (e.g., the one or more integration recognition sites or the one or more additional integration recognition sites). [0405] In certain embodiments, methods and compositions of the invention are adapted to differentiate muscle cells to cardiomyocytes for Duchene Muscular Dystrophy (DMD). The dystrophin gene is the largest gene in the human genome, spanning ∼2.3 Mb of DNA. DMD is composed of 79 exons resulting in a 14-kb full-length mRNA. Common mutations include mutations that disrupt the reading frame of generate a premature stop codon. An aspect of DMD that lends it to gene editing as a therapeutic approach is the modular structure of the dystrophin protein. Redundancy in the central rod domain permits the deletion of internal segments of the gene that may harbor loss-of-function mutations, thereby restoring the open reading frame (ORFs). [0406] The following are non-limiting diseases that may be treated utilizing the methods and compositions of the present disclosure: Inherited Retinal Diseases: • Stargardt Disease (ABCA4) • Leber congenital amaurosis 10 (CEP290) • X linked Retinitis Pigmentosa (RPGR) • Autosomal Dominant Retinitis Pigmentosa (RHO) Liver Diseases: • Wilson’s disease (ATP7B) • Alpha-1 antitrypsin (SERPINA1) Intellectual Disabilities: • Rett Syndrome (MECP2) • SYNGAP1-ID (SYNGAP1) • CDKL5 deficiency disorder (CDKL5) Peripheral Neuropathies: • Charcot-Marie-Tooth 2A (MFN2) Lung Diseases: • Cystic Fibrosis (CFTR) • Alpha-1 Antitrypsin (SERPINA1) Autoimmune diseases: • IgA Nephropathy (Berger’s disease) • Anti-Neutrophil Cytoplasmic Antibody (ANCA) Vasculitis • Systemic Lupus Erythematosus (SLE) / Lupus Nephritis (LN) • Membranous Nephropathy (MN) • C3 glomerulonephritis (C3GN) Blood disorders: • Sickle Cell • Hemophilia • Factor VIII or • Factor IX • Ornithine transcarbamylase deficiency (OTCD) • Homocystinuria (HCU) • Phenylketonuria (PKU) Cancer • Prostate cancer • Renal cell cancer • Thyroid cancer [0407] CFTR (cystic fibrosis transmembrane conductance regulator). The most common cystic fibrosis (CF) mutation F508del removes a single amino acid. In some embodiments, recombining human CFTR into an SHS of a cell that expresses CFTR F508del is a corrective treatment path. In some embodiments, the methods and systems described herein are used to CF by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing CF. Proposed validation is detection of persistent CFTR mRNA and protein expression in transduced cells. [0408] Sickle cell disease (SCD) is caused by mutation of a specific amino acid — valine to glutamic acid at amino acid position 6. In some embodiments, SCD is corrected by recombination of the HBB gene into a safe harbor site (SHS) and by demonstrating correction in a proportion of target cells that is high enough to produce a substantial benefit. In some embodiments, the methods and systems described herein are used to sickle cell disease by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the disease. In some embodiments, validation is detection of persistent HBB mRNA and protein expression in transduced cells. [0409] DMD — Duchenne Muscular Dystrophy. The dystrophin gene is the largest gene in the human genome, spanning ∼2.3 Mb of DNA. DMD is composed of 79 exons resulting in a 14- kb full-length mRNA. Common mutations include mutations that disrupt the reading frame of generate a premature stop codon. An aspect of DMD that lends it to gene editing as a therapeutic approach is the modular structure of the dystrophin protein. Redundancy in the central rod domain permits the deletion of internal segments of the gene that may harbor loss-of-function mutations, thereby restoring the open reading frame (ORFs). [0410] In some embodiments, recombination will be into safe harbor sites (SHS). A frequently used human SHS is the AAVS1 site on chromosome 19q, initially identified as a site for recurrent adeno-associated virus insertion. In some embodiments, the site is the human homolog of the e murine Rosa26 locus (pubmed.ncbi.nlm.nih.gov/18037879). In some embodiments, the site is the human H11 locus on chromosome 22. Proposed target cells for recombination include stem cells for example induced pluripotent stem cells (iPSCs) and cells at various stages of differentiation. In some cases, a complete gene may be prohibitively large and replacement of an entire gene impractical. In such instances, rescuing mutants by recombining in corrected gene fragments with the methods and systems described herein is a corrective option. [0411] In some embodiments, correcting mutations in exon 44 (or 51) by recombining in a corrective coding sequence downstream of exon 43 (or 50), using the methods and systems described herein is a corrective option. Proposed validation is detection of persistent DMD mRNA and protein expression in transduced cells. [0412] F8 (Factor VIII). A large proportion of severe hemophilia A patients harbor one of two types of chromosomal inversions in the FVIII gene. The recombinase technology and methods described herein are well suited to correcting such inversions (and other mutations) by recombining of the FVIII gene into a SHS. [0413] In some embodiments, correcting factor VIII deficiency by recombining the FVIII gene into an SHS is a corrective path. In some embodiments, the methods and systems described herein are used to correct factor VIII deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the FIX deficiency. Proposed validation is detection of persistent FVIII mRNA and protein expression in transduced cells. [0414] Factor 9 (Factor IX) Hemophilia B, also called factor IX (FIX) deficiency is a genetic disorder caused by missing or defective factor IX, a clotting protein. [0415] In some embodiments, the methods and systems described herein are used to correct factor IX deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the FIX deficiency. Proposed validation is detection of persistent FiX mRNA and protein expression in transduced cells. [0416] Ornithine transcarbamylase deficiency (OTCD). Ornithine transcarbamylase deficiency is a rare genetic condition that causes ammonia to build up in the blood. The condition – more commonly called OTC deficiency – is more common in boys than girls and tends to be more severe when symptoms emerge shortly after birth. [0417] In some embodiments, the methods and systems described herein are used to correct OTC deficiency by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the OTC deficiency or integrates a polynucleotide encoding a functional ornithine transcarbamylase enzyme. Proposed validation is detection of persistent OTC mRNA and protein expression in transduced cells. [0418] Phenylketonuria, also called PKU, is a rare inherited disorder that causes an amino acid called phenylalanine to build up in the body. PKU is caused by a change in the phenylalanine hydroxylase (PAH) gene. This gene helps create the enzyme needed to break down phenylalanine. [0419] In some embodiments, the methods and systems described herein are used to correct PKU by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the PKU deficiency or integrates a polynucleotide encoding a functional phenylalanine hydroxylase (PAH) gene. Proposed validation is detection of persistent PAH mRNA and protein expression in transduced cells. [0420] Homocystinuria (HCU). Homocystinuria is elevation of the amino acid, homocysteine (protein building block coming from our diet) in the urine or blood. Common causes of HCU include: problems with the enzyme cystathionine beta synthase (CBS), which converts homocysteine to the amino acid cystathionine (which then becomes cysteine) and needs the vitamin B6 (pyridoxine); and problems with converting homocysteine to the amino acid methionine. [0421] In some embodiments, the methods and systems described herein are used to correct HCU by site-specifically integrating in the genome a polynucleotide template that corrects the mutation causing the HCU or integrates a polynucleotide encoding a functional copy of a gene (e.g., CBS) able to reduce or prevent buildup of homocysteine in the urine. Proposed validation is detection of persistent CBS mRNA and protein expression in transduced cells. [0422] IgA Nephropathy (Berger’s disease). IgA nephropathy, also known as Berger's disease, is a kidney/autoimmune disease that occurs when an antibody called immunoglobulin A (IgA) builds up in the kidneys. [0423] In some embodiments, the methods and systems described herein are used to treat Berger’s disease by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein. Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of Berger’s disease. [0424] Anti-Neutrophil Cytoplasmic Antibody (ANCA) Vasculitis. ANCA vasculitis is an autoimmune disease affecting small blood vessels in the body. It is caused by autoantibodies called ANCAs, or Anti-Neutrophilic Cytoplasmic Autoantibodies. ANCAs target and attack a certain kind of white blood cells called neutrophils. [0425] In some embodiments, the methods and systems described herein are used to treat ANCA vasculitis by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein. Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of ANCA vasculitis. [0426] Systemic Lupus Erythematosus (SLE) / Lupus Nephritis (LN). Lupus is an autoimmune—a disorder in which the body’s immune system attacks the body’s own cells and organs. [0427] In some embodiments, the methods and systems described herein are used to treat SLE/LN by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein. Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of SLE/LN. [0428] Membranous Nephropathy (MN). MN is a kidney disease that affects the filters (glomeruli) of the kidney and can cause protein in the urine, as well as decreased kidney function and swelling. It can sometimes be called membranous glomerulopathy as well (these terms can be used interchangeably and mean the same thing). [0429] In some embodiments, the methods and systems described herein are used to treat MN by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein. Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of MN. [0430] C3 glomerulonephritis (C3GN). C3 glomerulopathy is a group of related conditions that cause the kidneys to malfunction. The major features of C3 glomerulopathy include high levels of protein in the urine (proteinuria), blood in the urine (hematuria), reduced amounts of urine, low levels of protein in the blood, and swelling in many areas of the body. Affected individuals may have particularly low levels of a protein called complement component 3 (or C3) in the blood. [0431] In some embodiments, the methods and systems described herein are used to treat C3 glomerulopathy by administering to a patient an iPSC-derived Natural Killer cell that includes a polynucleotide site-specifically integrated in the genome of the cell using the methods described herein. Upon administering the iPSC-NK cell to the patient, the iPSC-NK cell is capable of removing native cells (e.g., B cells) that are responsible, at least in part, for the symptoms of C3 glomerulopathy. 6.12. Pleiopluripotent Stem Cells [0432] In another embodiment, this disclosure features a pleiopluripotent cell comprising at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome. [0433] As used herein, the term “pleiopluripotent” refers to a pluripotent cell comprising at least a first integration recognition site. “Pluripotent” as used herein, refers to the ability of a cell to form all lineages of the body or the embryo proper. Pluripotency is a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell). “Pluripotent” as used herein is intended to include cells that are totipotent, i.e., cells that have the ability to develop into an entire organism. [0434] In another aspect, this disclosure features a donor polynucleotide template (e.g., any of the donor polynucleotide templates described herein) comprised of one or more orthogonal integration recognition site integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0435] In certain embodiments of any of the pleiopluripotent cells described herein, at least one of the at least first integration recognition site is incorporated into the pleiopluripotent cell genome at a safe harbor locus. [0436] In certain embodiments of any of the pleiopluripotent cells described herein, at least one of the at least first integration recognition sites is site-specifically incorporated within a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell. In some embodiments of any of the pleiopluripotent cells described herein, at least one of the at least first integration recognition sites is site-specifically incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof. In some embodiments of any of the pleiopluripotent cells described herein, the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ. In some embodiments of any of the pleiopluripotent cells described herein, the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus. In some embodiments of any of the pleiopluripotent cells described herein, the locus is the B2M locus. In some embodiments of any of the pleiopluripotent cells described herein, the locus is the CIITA locus. [0437] In some embodiments of any of the pleiopluripotent cells described herein, the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. [0438] In some embodiments of any of the pleiopluripotent cells described herein, the PC-derived cell or population thereof includes at least a first integration recognition site-specifically incorporated into the genome. In some embodiments of any of the pleiopluripotent cells described herein, at least one of the at least first integration recognition sites is specific for a serine integrase. In some embodiments of any of the pleiopluripotent cells described herein, at least one of the at least first integration recognition sites is an attB or attP site. In some embodiments of any of the pleiopluripotent cells described herein, at least one of the at least first integration recognition sites is a modified attB or attP site. In some embodiments of any of the pleiopluripotent cells described herein, at least one of the at least first integration recognition site is specific for BxB1 or a modified BxB1. In some of any of the pleiopluripotent cells described herein, at least one of the at least first integration recognition sites is comprised of 38 or 46 nucleotides. [0439] In some embodiments of any of the pleiopluripotent cells described herein, the pleiopluripotent cell includes a first donor polynucleotide (e.g., any of the donor polynucleotide templates described herein, see, for example, Section 4.11.3) integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, where the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0440] In some embodiments of any of the pleiopluripotent cells described herein, the pleiopluripotent cell includes a second integration recognition site (e.g., any of the integration recognition sites described herein) site-specifically incorporated into the pleiopluripotent cell genome. [0441] In some embodiments of any of the pleiopluripotent cells described herein, the second integration recognition sites is site-specifically incorporated into a safe harbor locus. [0442] In some embodiments of any of the pleiopluripotent cells described herein, the second integration recognition site is site-specifically incorporated within a second locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell. Non-limiting examples of loci where disruption is capable of reducing allogeneic immune response to the PC-derived cell are as described in Section 4.11.1. For example, without limitation, the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus. In some embodiments, the locus is the B2M locus. In some embodiments, the locus is the CIITA locus. In some embodiments, the locus where the first integration recognition site is incorporated into the genome and the second integration recognition site is incorporated into the genome are different. In some embodiments, the locus where the first integration recognition site is incorporated into the genome and the second integration recognition site is incorporated into the genome are the same. [0443] In some embodiments of any of the pleiopluripotent cells described herein, the PC-derived cell or population thereof includes a second integration recognition site-specifically incorporated into the genome. In some embodiments of any of the pleiopluripotent cells described herein, the second integration recognition sites is specific for a serine integrase. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition sites is an attB or attP site. In some embodiments of any of the pleiopluripotent cells described herein, the second integration recognition sites is a modified attB or attP site. In some embodiments of any of the pleiopluripotent cells described herein, the second integration recognition site is specific for BxB1 or a modified BxB1. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition sites is comprised of 38 or 46 nucleotides. [0444] In some embodiments of any of the pleiopluripotent cells described herein, the PC-derived cell or population thereof includes a second donor polynucleotide (e.g., any of the donor polynucleotide templates described herein, see, for example, Section 4.11.3) integrated into the pleiopluripotent stem cell genome at a second integration recognition site, where the second integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0445] In some embodiments of any of the pleiopluripotent cells described herein, the second donor polynucleotide template encodes one or more therapeutic agents. In some embodiments, the one or more therapeutic agents when expressed from the PC-derived cell or population thereof treats, ameliorates, or prevents a disease or condition in a subject. Non-limiting examples of therapeutic agents are as described in Sections 4.11.5 and 4.11.7. In some embodiments of any of the pleiopluripotent cells described herein, the one or more therapeutic agents is a HLA class I proteins. In some embodiments of any of the PC-derived cell or populations thereof described herein, the one or more therapeutic agents is a HLA class I proteins. [0446] In some embodiments of any of the pleiopluripotent cells described herein, the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents. In some embodiments of any of the pleiopluripotent cells described herein, the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agent. In some embodiments of any of the pleiopluripotent cells described herein, the second donor polynucleotide template comprises an inducible promoter operably linked to at least one therapeutic agent. [0447] In certain embodiments, the pleiopluripotent cells are derived from a mammal (e.g., a human, rhesus macaque (Macaca mulatta), bovine (Bos taurus), porcine (Sus scrofa), and mouse (Mus musculus)). In one embodiment, the pleiopluripotent cells are chimeric (i.e., a combination of iPSC derived from two or species). [0448] Non-limiting examples of pleiopluripotent cells include: induced pleiopluripotent cells (iPSC), embryonic stem cells (ESC), somatic cell nuclear transfer embryonic stem cells (ntES cells), epiblast stem cells (EpiSC) and parthenogenesis embryonic stem cell (pES cells). In certain embodiments, the pleiopluripotent cell is an embryonic stem cell or an induced pleiopluripotent cell. In one embodiment, pluripotent cells include cells capable of forming extraembryonic tissue (e.g., cell having totipotency). Pluripotency can be assessed using methods that include, but are not limited to: (i) pleiopluripotent cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pleiopluripotent cell markers (e.g., OCT4, NANOG, SOX2, SSEA3/4, SSEA5, TRA1-60/81, TRAl-85, TRA2-54); (iv) teratoma formation consisting of the three somatic lineages; (v) formation of embryoid bodies consisting of cells from the three somatic lineages ; and (vi) ability to differentiate to all three somatic lineages (ectoderm, mesoderm and endoderm). [0449] In certain embodiments, the pleiopluripotent cell is an induced pluripotent stem cell (iPSC) derived from a non-pluripotent stem cell. In such cases, the non-pluripotent stem cell can be reprogrammed into an induced pleiopluripotent cell. Non-pluripotent stem cell can be reprogrammed into a induced pluripotent stem cell using exogenous genetic material (e.g., overexpression of transcription factors (e.g., Oct4, Sox2, Klf4, and c-Myc)), exogenous non- genetic material (e.g., small molecules (e.g., (valproic acid (VPA), CHIR99021, 616452, tranylcypromine (VC6T), and Forskolin (FSK (VC6TF))), or a combination thereof. Non-limiting examples of reprogramming a non-pluripotent stem cell into a pluripotent stem cell include methods as described in PCT Publications WO 2022/072883A1, and WO 2022/006399A1; U.S. Patent Nos.10,428,310B2; 9,499,797B2; and 8,802,438B2, each of which are herein incorporated by reference in their entireties. [0450] Also provided herein are cell banks comprising any of the pleiopluripotent cells described herein. 6.13. Pleiopluripotent (PC)-derived cell or a population thereof [0451] In another embodiments, this disclosure provides a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site (e.g., any of the integration recognition sites described herein) is incorporated site-specifically into the PC- derived cell genome. [0452] In another embodiment, this disclosure provides a pleiopluripotent (PC)-derived cell or a population thereof (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. 6.13.1. PC-derived Hematopoietic Cell [0453] In some embodiments, a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a hematopoietic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site e.g., any of the integration recognition sites described herein) incorporated site-specifically into the PC-derived cell genome. [0454] In some embodiments, a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a hematopoietic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0455] In some embodiments, the hematopoietic cell is selected from: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells. 6.13.2. PC-derived Neuronal Cell [0456] In some embodiments, a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a neuronal cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site (e.g., any of the integration recognition sites described herein) incorporated site-specifically into the PSC-derived cell genome. [0457] In some embodiments, a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a neuronal cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0458] In some embodiments, the neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron. 6.13.3. PC-derived Cardiac Cell [0459] In some embodiments, a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a cardiac cell differentiated from a pleiopluripotent cell; and (ii) the PC- derived cell comprises at least a first integration recognition site (e.g., any of the integration recognition sites described herein) incorporated site-specifically into the PSC-derived cell genome. [0460] In some embodiments, a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a cardiac cell differentiated from a pleiopluripotent cell; and (ii) the PC- derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0461] In some embodiments, the cardiac cell is selected from: a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte. 6.13.4. PC-derived Pancreatic Cell [0462] In some embodiments, a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a pancreatic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site (e.g., any of the integration recognition sites described herein) incorporated site-specifically into the PC-derived cell genome. [0463] In some embodiments, a pleiopluripotent (PC)-derived cell or a population thereof where (i) the PC-derived cell is a pancreatic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0464] In some embodiments, the pancreatic cell is selected from a pancreatic progenitor cell, pancreatic endoderm, an endocrine pancreatic cell, an exocrine pancreatic cell, a islet progenitor, and a beta cell. 6.13.5. Features of PC-derived cell or Populations Thereof [0465] In some embodiments of any of the PC-derived cell or populations thereof described herein, the PC-derived cell further comprises two integration recognition sites, three integration recognition sites, four integration recognition sites, five integration recognition sites, six integration recognition sites, seven integration recognition sites, eight integration recognition sites, nine integration recognition sites, or ten or more integration recognition sites. [0466] In some embodiments of any of the PC-derived cell or populations thereof described herein, at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a safe harbor locus. [0467] In some embodiments of any of the PC-derived cell or populations thereof described herein, at least one of the at least first integration recognition sites is site-specifically incorporated within a locus, disruption of which is capable of reducing allogeneic immune response to the PC- derived cell. Non-limiting examples of loci where disruption is capable of reducing allogeneic immune response to the PC-derived cell are as described in Section 4.11.1. For example, without limitation, the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA- C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus. In some embodiments, the locus is the B2M locus. In some embodiments, the locus is the CIITA locus. [0468] In some embodiments of any of the PC-derived cell or populations thereof described herein, the PC-derived cell or population thereof includes at least a first integration recognition site-specifically incorporated into the genome. In some embodiments of any of the PC-derived cell or populations thereof described herein, at least one of the at least first integration recognition sites is specific for a serine integrase. In some embodiments of any of the PC-derived cell or populations thereof described herein, at least one of the at least first integration recognition sites is an attB or attP site. In some embodiments of any of the PC-derived cell or populations thereof described herein, at least one of the at least first integration recognition sites is a modified attB or attP site. In some embodiments of any of the PC-derived cell or populations thereof described herein, at least one of the at least first integration recognition site is specific for BxB1 or a modified BxB1. In some embodiments of any of the PC-derived cell or populations thereof described herein, at least one of the at least first integration recognition sites is comprised of 38 or 46 nucleotides. [0469] In some embodiments of any of the PC-derived cell or populations thereof described herein, the PC-derived cell or population thereof includes a first donor polynucleotide (e.g., any of the donor polynucleotide templates described herein, see, for example, Section 4.11.3) integrated into the pleiopluripotent stem cell genome at an at least first integration recognition site, where the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. 6.13.6. Second integration recognition site [0470] In some embodiments of any of the PC-derived cell or populations thereof described herein, the PC-derived cell or population thereof includes a second integration recognition site (e.g., any of the integration recognition sites described herein) site-specifically incorporated into the pleiopluripotent cell genome. [0471] In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition sites is site-specifically incorporated into a safe harbor locus. [0472] In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition site is site-specifically incorporated within a second locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent stem cell or a cell matured from the pleiopluripotent stem cell. Non-limiting examples of loci where disruption is capable of reducing allogeneic immune response to the PC- derived cell are as described in Section 4.11.1. For example, without limitation, the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus. In some embodiments, the locus is the B2M locus. In some embodiments, the locus is the CIITA locus. In some embodiments, the locus where the first integration recognition site is incorporated into the genome and the second integration recognition site is incorporated into the genome are different. In some embodiments, the locus where the first integration recognition site is incorporated into the genome and the second integration recognition site is incorporated into the genome are the same. [0473] In some embodiments of any of the PC-derived cell or populations thereof described herein, the PC-derived cell or population thereof includes a second integration recognition site- specifically incorporated into the genome. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition sites is specific for a serine integrase. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition sites is an attB or attP site. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition sites is a modified attB or attP site. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition site is specific for BxB1 or a modified BxB1. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second integration recognition sites is comprised of 38 or 46 nucleotides. [0474] In some embodiments of any of the PC-derived cell or populations thereof described herein, the PC-derived cell or population thereof includes a second donor polynucleotide (e.g., any of the donor polynucleotide templates described herein, see, for example, Section 4.11.3) integrated into the pleiopluripotent stem cell genome at a second integration recognition site, where the second integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome. [0475] In some embodiments of any of the PC-derived cell or populations thereof described herein, the second donor polynucleotide template encodes one or more therapeutic agents. In some embodiments, the one or more therapeutic agents when expressed from the PC-derived cell or population thereof treats, ameliorates, or prevents a disease or condition in a subject. Non-limiting examples of therapeutic agents are as described in Sections 4.11.5and 4.11.7. In some embodiments of any of the PC-derived cell or populations thereof described herein, the one or more therapeutic agents is a HLA class I proteins. In some embodiments of any of the PC-derived cell or populations thereof described herein, the one or more therapeutic agents is a HLA class I proteins. [0476] In some embodiments of any of the PC-derived cell or populations thereof described herein, the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agent. In some embodiments of any of the PC-derived cell or populations thereof described herein, the second donor polynucleotide template comprises an inducible promoter operably linked to at least one therapeutic agent. 6.14. Kits and Cell-based Compositions [0477] This disclosure also provides certain components or embodiments of cell-based products (e.g., genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell) or pharmaceutical compositions thereof, can be provided in a kit. For example, any of the cell-based products described herein (e.g., any of the genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a pancreatic cell) can be provided frozen and packaged as a kit, alone or along with separate containers of any of the other agents from the pre- conditioning or post-conditioning steps, and optional instructions for use. [0478] In some embodiments, the cell-based products (e.g., genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell) may be frozen (cryopreserved) before administration into a subject. In some embodiments, cell-based products (e.g., genetically modified pleiopluripotent cells or cells- derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell) may be frozen by suspending the cells in media containing at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 50%) human-derived serum and/or plasma, and lowering the temperature of the suspension to at least -80° C, thereby freezing the cell-based products. In some embodiments, the freezing media is approximately 30% human- derived serum and/or plasma and approximately 10% of an agent that prevents ice crystal formation during freezing, e.g., DMSO. In a further embodiment, the cell-based product (e.g., genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell) freezing media suspension is maintained at -80°C for at least 24 hours and then transferred to liquid nitrogen for the duration of the storage. In a further embodiment, the frozen cell-based product (e.g., genetically modified pleiopluripotent cells or cells-derived therefrom, such as a hematopoietic cell, a neuronal cell, a cardiac cell, or a beta cell) suspension is thawed at a temperature in the range of 34°C to 41°C. [0479] In some embodiments, the genetically modified pleiopluripotent cells are selected, isolated, or a combination thereof, prior to being banked (e.g., cryopreserved). In some embodiments, the genetically modified pleiopluripotent cells are selected in bulk before being banked (e.g., cryopreserved). In some embodiments, the genetically modified pleiopluripotent cells are isolated prior to being banked (e.g., cryopreserved). In such cases, the genetically modified pleiopluripotent cells are isolated and expanded prior to being banked (e.g., cryopreserved). [0480] In some embodiments, the genetically modified pluripotent stem cells are banked (e.g., cryopreserved) following incorporation of one or more integration recognition sites (e.g., any of the integration recognition sites described herein) but prior to integration of a donor polynucleotide template (e.g., any of the donor polynucleotide templates described herein). In such cases, the genetically modified pluripotent stem cells are thawed (and optionally allowed to recover) prior to integrating a donor polynucleotide template (e.g., any of the donor polynucleotide templates described herein) into the genome of the pluripotent stem cell. [0481] In some embodiments, the genetically modified pluripotent stem cells are banked (e.g., cryopreserved) following incorporation of one or more integration recognition sites (“beacons”) (e.g., any of the integration recognition sites described herein) and integration of a donor polynucleotide template (e.g., any of the donor polynucleotide templates described herein). In such cases, the genetically modified pluripotent stem cells can be thawed (and optionally allowed to recover) prior to integrating a second donor polynucleotide template (e.g., any of the donor polynucleotide templates described herein) into the genome of the pluripotent stem cell. In other cases, the genetically modified pluripotent stem cells can be thawed (and optionally allowed to recover) and used for downstream applications (e.g., differentiation to a hematopoietic stem cell, a neuronal cell, a cardiac cell, or a beta cell and use as a source for cell therapy). [0482] In some embodiments, the cells (e.g., genetically modified pluripotent stem cells) are frozen, which can be referred to as cryopreservation. The cells (e.g., genetically modified pluripotent stem cells) can, for example, be frozen using CryoStor CS5 (freeze media), Cryostor CS10 (freeze media), or another freeze media. However, this is a non-limiting example of freezing media and other freezing media can be used. The cells (e.g., genetically modified pluripotent stem cells) can be initially chilled 4°C. Then the cells (e.g., genetically modified pluripotent stem cells) can be further cooled to about -20°C. The cells (e.g., genetically modified pluripotent stem cells) can be centrifuged or resuspended in additional freeze media and then cooled to -90°C in a stepwise manner. The cells (e.g., genetically modified pluripotent stem cells) can then be stored in liquid nitrogen. In some embodiments, the cells (e.g., genetically modified pluripotent stem cells) are frozen in a cryobag. [0483] In some embodiments, the composition may comprise a pharmaceutically acceptable excipient, a pharmaceutically acceptable salt, diluents, carriers, vehicles and such other inactive agents well known to the skilled artisan. Vehicles and excipients commonly employed in pharmaceutical preparations include, for example, talc, gum Arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, etc. Solutions can be prepared using water or physiologically compatible organic solvents such as ethanol, 1,2-propylene glycol, polyglycols, dimethylsulfoxide, fatty alcohols, triglycerides, partial esters of glycerine and the like. Compositions may be prepared using conventional techniques that may include sterile isotonic saline, water, 1,3-butanediol, ethanol, 1,2-propylene glycol, polyglycols mixed with water, Ringer's solution, etc. In one aspect, a coloring agent is added to facilitate in locating and properly placing the composition to the intended treatment site. [0484] Compositions may include a preservative and/or a stabilizer. Non-limiting examples of preservatives include methyl-, ethyl-, propyl-parabens, sodium benzoate, benzoic acid, sorbic acid, potassium sorbate, propionic acid, benzalkonium chloride, benzyl alcohol, thimerosal, phenylmercurate salts, chlorhexidine, phenol, 3-cresol, quaternary ammonium compounds (QACs), chlorbutanol, 2-ethoxyethanol, and imidurea. [0485] To control tonicity, the composition can comprise a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride and calcium chloride. [0486] Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included at a concentration in the 5-20 mM range. The pH of a composition will generally be between 5 and 8, and more typically between 6 and 8 e.g. between 6.5 and 7.5, or between 7.0 and 7.8. [0487] In some embodiments, the composition may include a cryoprotectant agent. Non-limiting examples of cryoprotectant agents include a glycol (e.g., ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, and any combinations thereof. [0488] Some embodiments are also directed to any of the aforementioned compositions in a kit. In some embodiments, the kit may comprise ampoules, disposable syringes, capsules, vials, tubes, or the like. In some embodiments, the kit may comprise a single dose container or multiple dose containers comprising the topical formulation of embodiments herein. In some embodiments, each dose container may contain one or more unit doses. In some embodiments, the kit may include an applicator. In some embodiments, the kits include all components needed for the stages of conditioning/treatment. In some embodiments, the cellular compositions may have preservatives or be preservative-free (for example, in a single-use container). In some embodiments, the cell-based products may be prepared and frozen at a desired stage, suitable for shipping to a hospital or treatment center. [0489] Also provided herein are methods for freezing, thawing, and expanding any of the pluripotent stem cells described herein. 6.15. Pharmaceutical Compositions [0490] This disclosure provides pharmaceutical compositions comprising the pluripotent stem cell or hematopoietic cell, cardiac cell or neuronal cell derived from the pluripotent stem cell, wherein the pharmaceutical compositions further comprise a pharmaceutically acceptable medium. In one embodiment, the pharmaceutical composition comprises the pluripotent cell derived hematopoietic cells made by the methods and composition disclosed herein. In one embodiment, the pharmaceutical composition comprises the pluripotent cell derived cardiac cells made by the methods and composition disclosed herein. In one embodiment, the pharmaceutical composition comprises the pluripotent cell derived neuronal cells made by the methods and composition disclosed herein. 6.16. Directed Differentiation of Pleiopluripotent Cells [0491] In certain embodiments, the method further includes directing differentiation of the modified pluripotent stem cell to a hematopoietic cell. Non-limiting examples of a hematopoietic cell include: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, platelets, erythrocytes, erythrocyte progenitors, megakaryocytes, and megakaryocyte precursors. Non-limiting methods of directing differentiation of the modified pluripotent stem cell to a hematopoietic cell include the methods described in U.S. Patent Pub. Nos. 2019/0119643A1; 2019/0177695A1, 2020/0199535A1, U.S. Patent Nos.10,858,628; 11,162,075; 10,947,505; and 11,162,076, each of which are herein incorporated by reference in their entireties. [0492] In certain embodiments, the method further includes directing differentiation of the modified pluripotent stem cell to a neuronal cell. Non-limiting examples of neuronal cells include: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron. Non-limiting methods of directing differentiation of the modified pluripotent stem cell to a neuronal cell include the methods described in the PCT Publication Nos. WO2022/134031A1; WO2022/136306A1; WO2022/093375A1; WO2022/014681A1; and WO2022005023A1; U.S. Patent Nos. 11,007,232; 10,519,421; and 10,487,311; and U.S. Patent Pubs.2021/0244768A1, each of which are herein incorporated by reference in their entireties. [0493] In certain embodiments, the method further comprises directing differentiation of the modified pluripotent stem cell to a cardiac cell. Non-limiting examples of a cardiac cell include: a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte. Non-limiting methods of directing differentiation of the modified pluripotent stem cell to a cardiomyocyte include the methods described in PCT Publication Nos. WO2020/247957; WO2021/187601A1; WO2021/172542A1; and WO2020/264308A1; and U.S. Patent Nos. 11,352,604; 10,973,876; 10,443,044; and 9,587,220; and U.S. Patent Pubs.2021/0363487A1, and 2020/0140819A1, each of which are herein incorporated by reference in their entireties. [0494] In certain embodiments, the method further comprises directing differentiation of the modified pluripotent stem cell to a pancreatic cell. Non-limiting examples of a pancreatic cell include: a pancreatic progenitor cell, pancreatic endoderm, an endocrine pancreatic cell, an exocrine pancreatic cell, a islet progenitor, and a beta cell. Non-limiting methods of directing differentiation of the modified pluripotent stem cell to a beta cell include the methods described in PCT Publication Nos. WO2020/033879A1; WO2021/030424A1; and WO2020/264072A1, U.S. Patent Nos. 10,376,545; and 9,650,610; U.S. Patent Publication No. US20210198632A1, each of which are herein incorporated by reference in their entireties. 6.17. Method of Using the Pleiopluripotent Cell (PC) or PC-derived Cell [0495] This disclosure also provides a method of using a pleiopluripotent cell having at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome, the method comprising: integrating a first donor polynucleotide template into the pleiopluripotent cell genome by introducing into the pleiopluripotent cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent stem cell at the at least one incorporated genomic integration recognition sites by the integrase. [0496] In some embodiments, the method of using pleiopluripotent cells also includes selecting the pleiopluripotent stem cells having the first donor polynucleotide template site-specifically integrated into the genome. In some embodiments, the method of using pleiopluripotent cells also includes expanding the pleiopluripotent cells in a de-differentiated state. [0497] In some embodiments, the method of using pleiopluripotent cells also includes cryopreserving the pleiopluripotent cells following incorporation of the at least first integration recognition site. In some embodiments, the method of using pleiopluripotent cells also includes cryopreserving the pleiopluripotent cells following integrating the donor polynucleotide template at the at least one incorporated genomic recognition site. [0498] In some embodiments, the method of using the pleiopluripotent cells also includes directing differentiation of the modified pleiopluripotent cell to a hematopoietic cell. Non- limiting examples of hematopoietic cells include: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells. [0499] In some embodiments, the method of using the pleiopluripotent cells also includes directing differentiation of the modified pleiopluripotent cell to a neuronal cell. Non-limiting examples of a neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron. [0500] In some embodiments, the method of using the pleiopluripotent cells also includes directing differentiation of the modified pleiopluripotent cell to a cardiac cell. Non-limiting examples of cardiac cells include a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte. [0501] In some embodiments, the method of using the pleiopluripotent cells also includes directing differentiation of the modified pleiopluripotent cell to a pancreatic cell. Non-limiting examples of pancreatic cells include pancreatic progenitor cell, pancreatic endoderm, an endocrine pancreatic cell, an exocrine pancreatic cell, an islet progenitor, and a beta cell. [0502] In some embodiments, the method of using the pleiopluripotent cells also includes administering the PC-derived cells or a population thereof to a subject (patient) in need thereof. 6.18. Programmable Gene Insertion for Research Purposes [0503] In another aspect, the methods and compositions described herein can be used to generate cell lines for research purposes or applications that are intended for research purposes only. For example, the methods (see, e.g., Section 6.11), cells (e.g., Section 6.12 and 6.13), kits (see, e.g., Section 6.14), and compositions (see, e.g., Section 6.15) can be used for research purposes or applications that are intended for research purposes only. In one embodiment, the methods (see, e.g., Section 6.11), cells (e.g., Section 6.12 and 6.13), kits (see, e.g., Section 6.14), and compositions (see, e.g., Section 6.15) can be used for screening purposes, for example, screening therapeutic molecules (e.g., compounds, small molecules, and large molecules). 6.19. Methods of Treatment [0504] In another aspect, methods of treatment are presented. In some embodiments, the subject has a disease, condition, and/or an injury that can be treated, ameliorated, and/or improved by a cell therapy. Some embodiments contemplate that a subject in need of cell therapy is a subject with an injury, disease, or condition, whereby a cell therapy, for example, a therapy in which a cellular material is administered to the subject, can treat, ameliorate, improve, and/or reduce the severity of at least one symptom associated with the injury, disease, or condition. [0505] Certain embodiments contemplate that a subject in need of cell therapy, includes, but is not limited to, a candidate for bone marrow or stem cell transplantation, a subject who has received chemotherapy or irradiation therapy, a subject who has or is at risk of having a hyperproliferative disorder or a cancer, (e.g., a hyperproliferative disorder) or a cancer of hematopoietic system, a subject having or at risk of developing a tumor (e.g., a solid tumor), a subject who has or is at risk of having a viral infection or a disease associated with a viral infection. [0506] This disclosure also features a method of treating or ameliorating or preventing a disease or condition in a subject, comprising administering a therapeutically effective amount of any of the PC-derived cell or population thereof, compositions, or pharmaceutical composition described herein. [0507] In some embodiments, the method includes treating or ameliorating or preventing cancer, where the subject has, or is suspected of having, cancer. [0508] In some embodiments, the method includes treating or ameliorating or preventing a cardiac condition, where the subject has or is suspected of having, a cardiac condition. In such cases, the cardiac condition is muscular and/or the condition is muscle degeneration or muscle injury. [0509] In some embodiments, the method includes treating or ameliorating or preventing a neuronal-associated disease, wherein the subject has, or is suspected of having, a neuronal- associated disease. In such cases, the disease is a neuronal-associated disease and/or the condition is neuron degeneration. [0510] In some embodiments, the method includes treating or ameliorating or preventing a pancreatic-associated disease, wherein the subject has, or is suspected of having, a pancreatic- associated disease. [0511] In some embodiments, the method of treating further includes administering to the subject, having previously been administered any of pluripotent stem cells described herein, one or more exogenous molecules to control activity of the one or more inducible suicide genes. In a non- limiting example, the method of treating also includes, following administration of any of the PC- derived cells or populations thereof as described herein, administering AP20187 (or an analog thereof), AP21967, ganciclovir, or a combination thereof. [0512] In some embodiments, the method of cell delivery used here occurs using electroporation. [0513] In some embodiments, the method of cell delivery used here occurs using a recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, anellovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, mRNA, RNP, or lipid nanoparticle. Delivery of the nucleic acid construct can also be by fusosome or exosome, (See, e.g., WO2019222403 which is incorporated by reference herein). Delivery of nucleic acid construct can also be by VesiCas (See, e.g., US20210261957A1 which is incorporated by reference herein). [0514] Methods of non-viral delivery include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). 6.19.1. Viral Vector Delivery into the Pluripotent Stem Cell Genome [0515] In certain aspects the invention involves vectors, e.g. for delivering or introducing in a cell, but also for propagating these components (e.g. in prokaryotic cells). A used herein, a "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double- stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors." Vectors for and that result in expression in a eukaryotic cell can be referred to herein as "eukaryotic expression vectors." Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. [0516] Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). With regards to recombination and cloning methods, mention is made of U.S. patent application Ser. No.10/815,730, published Sep.2, 2004 as US 2004-0171156 A1, the contents of which are herein incorporated by reference in their entirety. [0517] Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, for instance a Type V protein such as C2c1 or C2c3, and/or any of the present RNAs, for instance a guide RNA, can be delivered using any suitable vector, e.g., plasmid or viral vectors, such as adeno associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof. Effector proteins and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmid or viral vectors. [0518] The promoter used to drive nucleic acid-targeting effector protein coding nucleic acid molecule expression can include: AAV ITR can serve as a promoter: this is advantageous for eliminating the need for an additional promoter element (which can take up space in the vector). The additional space freed up can be used to drive the expression of additional elements (gRNA, etc.). Also, ITR activity is relatively weaker, so can be used to reduce potential toxicity due to over expression of nucleic acid-targeting effector protein. For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc. For brain or other CNS expression, can use promoters: Synapsin I for all neurons, CaMKII alpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For liver expression, can use Albumin promoter. For lung expression, can use SP-B. For endothelial cells, can use ICAM. For hematopoietic cells can use IFNbeta or CD45. For Osteoblasts can use OG-2. [0519] The promoter used to drive guide RNA can include: Pol III promoters such as U6 or H1 Use of Pol II promoter and intronic cassettes to express guide RNA Adeno Associated Virus (AAV) [0520] Nucleic acid-targeting effector protein and one or more guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Pat. No.8,454,972 (formulations, doses for adenovirus), U.S. Pat. No.8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus. For examples, for AAV, the route of administration, formulation and dose can be as in U.S. Pat. No. 8,454,972 and as in clinical trials involving AAV. For Adenovirus, the route of administration, formulation and dose can be as in U.S. Pat. No. 8,404,658 and as in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as in U.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids. Doses may be based on or extrapolated to an average 70 kg individual (e.g., a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed. The viral vectors can be injected into the tissue of interest. For cell- type specific genome modification, the expression of nucleic acid-targeting effector can be driven by a cell-type specific promoter. For example, liver-specific expression might use the Albumin promoter and neuron-specific expression (e.g., for targeting CNS disorders) might use the Synapsin I promoter. [0521] Another useful method to deliver proteins, enzymes, and guides to a pluripotent stem cell comprises transfection of messenger RNA (mRNA). Examples of mRNA delivery methods and compositions that may be utilized in the present disclosure including, for example, PCT/US2014/028330, US8822663B2, NZ700688A, ES2740248T3, EP2755693A4, EP2755986A4, WO2014152940A1, EP3450553B1, BR112016030852A2, and EP3362461A1. Expression of CRISPR systems in particular is described by WO2020014577. Each of these publications are incorporated herein by reference in their entireties. Additional disclosure hereby incorporated by reference can be found in Kowalski et al., “Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery,” Mol Therap., 2019; 27(4): 710-728. [0522] In some embodiments, one or more vectors described herein are used to produce a non- human transgenic animal or transgenic plant. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, or rabbit. In certain embodiments, the organism or subject is a plant. In certain embodiments, the organism or subject or plant is algae. Methods for producing transgenic plants and animals are known in the art, and generally begin with a method of cell transfection, such as described herein. [0523] In some embodiments, the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including micro-algae) and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant (including micro-algae). [0524] Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A "disease-associated" gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown and may be at a normal or abnormal level. [0525] In some embodiments, the genetically modified cell are non-dividing cells (e.g., where cell growth has been arrested) prior to being contacted with the gene editing reagents (e.g., gene editor protein or a polynucleotide encoding a gene editor protein, one or more guide RNA, and optionally a nicking gRNA). In some embodiments, the genetically modified cells are terminally differentiated, non-dividing cells prior to being contacted with the gene editing reagents (e.g., gene editor protein or a polynucleotide encoding a gene editor protein, one or more guide RNA, and optionally a nicking gRNA). In some embodiments, the genetically modified cells are dividing cells. 7. ADDITIONAL EMBODIMENTS [0526] Embodiment 1. A method of editing a pluripotent stem cell, the method comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the one or more integration target recognition sites into the genome; and (ii) two guide RNAs, wherein each of the two guide RNA encodes all or part of an integrase target recognition site. [0527] Embodiment 2. A method of editing a pluripotent stem cell, the method comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the one or more integration target recognition sites into the genome; and (ii) two guide RNA complexes, wherein each of the two guide RNA complexes is comprised of at least one polynucleotide that encodes all or part of an integrase target recognition site. [0528] Embodiment 3. A method of editing a pluripotent stem cell, the method comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the one or more integration target recognition sites into the genome; (ii) one or more guide RNA, wherein the one or more guide RNA encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA. [0529] Embodiment 4. A method of editing a pluripotent stem cell, the method comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of polynucleotide genomic integration; (ii) one or more guide RNA complex, wherein the one or more guide RNA complex is comprised of at least one polynucleotide that encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA. [0530] Embodiment 5. The method of any one of embodiments 1-4, further comprising: b) integrating a donor polynucleotide template into the pluripotent stem cell genome at the one or more incorporated target recognition sites by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration target recognition sites, wherein the donor polynucleotide is integrated into the pluripotent stem cell at the one or more incorporated genomic integration target recognition sites by an integrase; thereby producing a genetically modified pluripotent stem cell. [0531] Embodiment 6. A method of editing a pluripotent stem cell, the method comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the one or more integration target recognition sites into the genome; and (ii) two guide RNAs, wherein each of the two guide RNA encodes all or a portion of an integrase target recognition site; and (b) integrating a donor polynucleotide template into the pluripotent stem cell genome at the one or more incorporated target recognition sites by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration target recognition sites, wherein the donor polynucleotide is integrated into the pluripotent stem cell at the incorporated genomic integration target recognition site by an integrase; thereby producing a genetically modified pluripotent stem cell. [0532] Embodiment 7. A method of editing a pluripotent stem cell genome, the method comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of polynucleotide genomic integration; and (ii) two guide RNA complexes, wherein each of the two guide RNA complexes is comprised of at least one polynucleotide that encodes all or part of an integrase target recognition site; and (b) integrating a donor polynucleotide template into the pluripotent stem cell genome at the one or more incorporated target recognition sites by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one of more integration target recognition sites, wherein the donor polynucleotide is integrated into the pluripotent stem cell at the one or more incorporated genomic integration target recognition sites by an integrase; thereby producing a genetically modified pluripotent stem cell. [0533] Embodiment 8. A method of editing a pluripotent stem cell, the method comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the one or more integration target recognition sites into the genome; (ii) one or more guide RNA, wherein the one or more guide RNA encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA; and (b) integrating a donor polynucleotide template into the pluripotent stem cell genome at the one or more incorporated target recognition sites by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration target recognition sites, wherein the donor polynucleotide is integrated into the pluripotent stem cell at the incorporated genomic integration target recognition site by an integrase; thereby producing a genetically modified pluripotent stem cell. [0534] Embodiment 9. A method of editing a pluripotent stem cell genome, the method comprising: (a) incorporating one or more integration target recognition sites into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of polynucleotide genomic integration; (ii) one or more guide RNA complex, wherein the one or more guide RNA complex is comprised of at least one polynucleotide that encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA; and (b) integrating a donor polynucleotide template into the pluripotent stem cell genome at the one or more incorporated target recognition sites by delivering into the cell: (i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one of more integration target recognition sites, wherein the donor polynucleotide is integrated into the pluripotent stem cell at the one or more incorporated genomic integration target recognition sites by an integrase; thereby producing a genetically modified pluripotent stem cell. [0535] Embodiment 10. The method of any one of embodiments 6-9, wherein steps (a) and (b) are performed concurrently. [0536] Embodiment 11. The method of any one of embodiments 1-10, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. [0537] Embodiment 12. The method of any one of embodiments 1-11, wherein the one or more integration target recognition sites is incorporated at one or more loci associated with HLA Class I proteins, one or more loci associated with HLA Class II proteins, or a combination thereof. [0538] Embodiment 13. The method of embodiment 12, wherein the one or more integration target recognition sites is incorporated at one or more of the B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus. [0539] Embodiment 14. The method of any one of embodiments 1-13, wherein the pluripotent stem cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. [0540] Embodiment 15. The method of embodiment 14, wherein the one or more HLA Class I proteins, one or more HLA Class II proteins are encoded in one or more chromosomal regions selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ. [0541] Embodiment 16. The method of embodiment 14, wherein the one or more HLA Class I proteins or one or more HLA Class II proteins are selected from: B2M, and CIITA. [0542] Embodiment 17. The method of any one of embodiments 1-16, wherein delivery is performed by electroporation. [0543] Embodiment 18. The method of any one of embodiments 1-17, wherein delivery into the pluripotent stem cell occurs using one or more of a recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, anellovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, mRNA, RNP, or lipid nanoparticle. [0544] Embodiment 19. The metho of any one of embodiments 1-16, wherein delivery is performed by transfection. [0545] Embodiment 20. The method of embodiment 19, wherein delivery comprises transfecting mRNA encoding the gene editor protein, the one or more guide gRNA or two guide RNAs, and the donor polynucleotide template, wherein the donor polynucleotide template is selected from a mini circle, a nanoplasmid, and a miniDNA. [0546] Embodiment 21. The method of any one of embodiments 1-20, wherein the one or more integration target recognition sites is specific for a serine integrase. [0547] Embodiment 22. The method of any one of embodiments 1-21, wherein the one or more integration target recognition sites is an attB or attP site. [0548] Embodiment 23. The method of any one of embodiments 1-21, wherein the one or more integration target recognition sites is a modified attB or attP site. [0549] Embodiment 24. The method of any one of embodiments 1-23, wherein the one or more integration target recognition sites is specific for BxB1 or a modified BxB1. [0550] Embodiment 25. The method of any one of embodiments 1-24, wherein the one or more integration target recognition sites is comprised of 38 or 46 nucleotides. [0551] Embodiment 26. The method of any one of embodiments 1-25, wherein the one or more guide RNA or the one or more guide RNA complex comprises a chemical modification. [0552] Embodiment 27. The method of any one of embodiments 1-26, wherein the donor polynucleotide template encodes for one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof. [0553] Embodiment 28. The method of embodiment 27, wherein expression of the one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof, is driven by a pluripotent stem cell-specific promoter. [0554] Embodiment 29. The method of embodiment 27, wherein expression of the one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof, is driven by a constitutive promoter. [0555] Embodiment 30. The method of any one of embodiments 27-29, wherein the one or more inducible suicide genes is selected from: caspase9, cytosine deasminase, and thymidine kinase. [0556] Embodiment 31. The method of embodiment 30, wherein the one or more inducible suicide genes is a controllable caspase9. [0557] Embodiment 32. The method of embodiment 31, wherein AP20187 (or an analog thereof) controls activity of Caspase9 or AP21967 (or an analog thereof) controls activity of Caspase9. [0558] Embodiment 33. The method of embodiment 31, further comprising a second inducible suicide gene, wherein the second inducible suicide gene comprises a thymidine kinase. [0559] Embodiment 34. The method of embodiment 33, wherein the polynucleotide encoding for the thymidine kinase is operably linked to the controllable capase9 protein using a 2A self-cleaving peptide or an internal ribosome entry site. [0560] Embodiment 35. The method of any one of embodiments 1-34, wherein the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL- 1, B2M-HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof. [0561] Embodiment 36. The method of embodiment 35, wherein the donor polynucleotide template encodes a CD47, PDL1, and B2M-HLA-E. [0562] Embodiment 37. The method of embodiment 36, wherein the polynucleotide template encoding CD47, PDL1 and B2M-HLA-E are separated by a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof. [0563] Embodiment 38. The method of any one of embodiments 27-37, wherein the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag. [0564] Embodiment 39. The method of any one of embodiments 27-38, wherein the donor polynucleotide template that encodes one or more inducible suicide genes further comprises an integrase target recognition site. [0565] Embodiment 40. The method of any one of embodiments 27-39, wherein the donor polynucleotide template that encodes one or more exogenous polypeptides capable of modulating an immune response further comprises an integrase target recognition site. [0566] Embodiment 41. The method of any one of embodiments 1-40, further comprising: incorporating an additional integration target recognition site into the pluripotent stem cell genome by delivering into the cell: (i) a gene editor protein or polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of polynucleotide genomic integration; (ii) one or more guide RNA or one or more guide RNA complex, wherein the one or more guide RNA or one or more guide RNA complex is comprised of at least one polynucleotide that encodes an integrase target recognition site; and (iii) optionally, a nicking gRNA. [0567] Embodiment 42. The method of embodiment 41, further comprising: integrating a second donor polynucleotide template into the pluripotent stem cell genome at the incorporated additional target recognition site by delivering into the cell: (i) the second donor polynucleotide template, wherein the second donor polynucleotide template is comprised of an additional integration target recognition site, wherein the second donor polynucleotide is integrated into the pluripotent stem cell at the incorporated additional genomic integration target recognition site by an integrase; thereby producing a genetically modified pluripotent stem cell comprising a first polynucleotide template and a second polynucleotide template each integrated into the pluripotent stem cell genome. [0568] Embodiment 43. The method of any one of embodiments 1-42, further comprising directing differentiation of the modified pluripotent stem cell to a hematopoietic cell. [0569] Embodiment 44. The method of embodiment 43, wherein the hematopoietic cell is selected from: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells. [0570] Embodiment 45. The method of any one of embodiments 1-42, further comprising directing differentiation of the modified pluripotent stem cell to a neuronal cell. [0571] Embodiment 46. The method of embodiments 45, wherein the neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron. [0572] Embodiment 47. The method of any one of embodiments 1-42, further comprising directing differentiation of the modified pluripotent stem cell to a cardiac cell. [0573] Embodiment 48. The method of embodiment 47, wherein the cardiac cell is selected from: a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte. [0574] Embodiment 49. A pluripotent stem cell comprising: (a) an integration target recognition site integrated in the pluripotent stem cell genome at one or more loci associated with HLA Class I proteins, one or more loci associated with HLA Class II proteins, or a combination thereof. [0575] Embodiment 50. A pluripotent stem cell comprising: (a) a donor polynucleotide template integrated into the pluripotent stem cell genome at one or more loci associated with HLA Class I proteins, one or more loci associated with HLA Class II proteins, or a combination thereof. [0576] Embodiment 51. A pluripotent stem cell comprising: (a) an integration target recognition site that is integrated (or capable of being integrated) in the pluripotent stem cell genome at one or more loci associated with HLA Class I proteins, one or more loci associated with HLA Class II proteins, or a combination thereof; (b) a gene editor protein or a polynucleotide encoding a gene editor protein, wherein the gene editor protein is capable of incorporating the integration target recognition site into the genome; (c) one or more guide RNA, wherein the one or more guide RNA encodes an integrase target recognition site; and (d) optionally, a nicking gRNA. [0577] Embodiment 52. The pluripotent stem cell of embodiment 51, further comprising: (e) a donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration target recognition site, wherein the donor polynucleotide is integrated into the pluripotent stem cell genome at the incorporated genomic integration target recognition site. [0578] Embodiment 53. The pluripotent stem cell of any one of embodiments 49-52, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. [0579] Embodiment 54. The pluripotent stem cell of any one of embodiments 49-53, wherein the one or more integration target recognition sites is incorporated at one or more of the B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA- DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus. [0580] Embodiment 55. The pluripotent stem cell of any one of embodiments 49-54, wherein the donor polynucleotide template is incorporated at one or more of the B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus. [0581] Embodiment 56. The pluripotent stem cell of any one of embodiments 49-55 wherein the pluripotent stem cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof. [0582] Embodiment 57. The pluripotent stem cell of embodiment 56, wherein the one or more HLA Class I proteins or one or more HLA Class II proteins are selected from: B2M and CIITA. [0583] Embodiment 58. The pluripotent stem cell of any one of embodiments 49-57, wherein the integration target recognition site is an attB or attP site. [0584] Embodiment 59. The pluripotent stem cell of any one of embodiments 49-58, wherein the integration target recognition site is a modified attB or attP site. [0585] Embodiment 60. The pluripotent stem cell of any one of embodiments 49-59, wherein the integration target recognition site is specific for BxB1 or a modified BxB1. [0586] Embodiment 61. The pluripotent stem cell of any one of embodiments 49-60, wherein the integration target recognition site is comprised of 38 or 46 nucleotides. [0587] Embodiment 62. The pluripotent stem cell of any one of embodiments 49-61, wherein the one or more guide RNA or the one or more guide RNA complex comprises a chemical modification. [0588] Embodiment 63. The pluripotent stem cell of any one of embodiments 49-62, wherein the donor polynucleotide template encodes for one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof. [0589] Embodiment 64. The pluripotent stem cell of embodiment 63, wherein expression of the one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof, is driven by a pluripotent stem cell-specific promoter. [0590] Embodiment 65. The pluripotent stem cell of embodiment 63, wherein expression of the one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof, is driven by a constitutive promoter. [0591] Embodiment 66. The pluripotent stem cell of any one of embodiments 63-65, wherein the one or more inducible suicide genes is select from: caspase9, cytosine deasminase, and thymidine kinase. [0592] Embodiment 67. The pluripotent stem cell of embodiment 66, wherein the one or more inducible suicide genes is a controllable caspase9. [0593] Embodiment 68. The pluripotent stem cell of embodiment 67, wherein AP20187 (or analog thereof) controls activity of Caspase9 or AP21967 (or analog thereof) controls activity of Caspase9. [0594] Embodiment 69. The pluripotent stem cell of embodiment 67, further comprising a second inducible suicide gene, wherein the second inducible suicide gene comprises a thymidine kinase. [0595] Embodiment 70. The pluripotent stem cell of embodiment 69, wherein the polynucleotide encoding for the thymidine kinase is operably linked to the controllable capase9 protein using a 2A self-cleaving peptide or an internal ribosome entry site. [0596] Embodiment 71. The pluripotent stem cell of any one of embodiments 63-70, wherein the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL-1, B2M-HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof. [0597] Embodiment 72. The pluripotent stem cell of embodiment 71, wherein the donor polynucleotide template encodes a CD47, PDL1, and B2M-HLA-E. [0598] Embodiment 73. The pluripotent stem cell of embodiment 72, wherein the polynucleotide template encoding CD47, PDL1 and B2M-HLA-E are separated by a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof. [0599] Embodiment 74. The pluripotent stem cell of any one of embodiments 63-73, wherein the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag. [0600] Embodiment 75. The pluripotent stem cell of embodiments 63-71, wherein the one or more inducible suicide genes further comprise an integrase target recognition site. [0601] Embodiment 76. The pluripotent stem cell of any one of embodiments 63-75, wherein the one or more exogenous polypeptides capable of modulating an immune response further comprises an integrase target recognition site. [0602] Embodiment 77. A composition comprising the pluripotent stem cell of embodiments 49- 76. A pharmaceutical composition one or more of the pluripotent stem cells of claims 49-76 and a pharmaceutically acceptable excipient. 8. EXAMPLES 8.1. Materials [0603] Primers and probes used in ddPCR experiments described in the Examples can be found in Table 13.
Figure imgf000237_0001
Table 13. ddPCR Primers and Probes
Figure imgf000238_0001
8.2. Example 1: Dual atgRNA-mediated beacon placement within the B2M or CIITA loci in iPSC [0604] To demonstrate and optimize attB beacon placement (i.e., incorporation into the genome of the integrase target recognition site) at the B2M locus or the CIITA locus within iPSCs, a dual atgRNA approach to beacon placement was used. A non-limiting example of a dual atgRNA approach for beacon placement within an iPSC genome is as shown in FIG.6 (left panel showing example atgRNAs and right panel showing a schematic of the dual atgRNA approach). The dual atgRNAs (forward and reverse atgRNAs) each included a portion of the 38 bp attB insertion site within a segment of the reverse transcriptase template (see, e.g., FIGs.7A-7B) (Table 12). The protospacer sequences (i.e., a sequence/domain capable of guiding the gene editor protein or prime editor fusion protein to site-specifically nick the iPSC genome) were initially selected from the literature (see FIG.6 and FIG.9). [0605] A non-limiting example of a method of generating an iPSC having an integration recognition site site-specifically integrated into the iPSC genome is shown in FIG.8. [0606] iPSC lines ACS-1030 (iPSC1) and ACS-1026 (iPSC2) were purchased from ATCC and cultured as undifferentiated iPSCs grown on Cell Basement Membrane (ATCC)-coated plates in Pluripotent Stem Cell SFM XF/FF media (ATCC). Undifferentiated iPSCs were electroporated with prime editor fusion protein (2 micrograms mRNA encoding prime editor fusion protein) and atgRNA (50 picomoles). Electroporation was performed using a Neon Transfection System (ThermoFisher) with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane-coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM Rho Kinase (ROCK) inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, media was changed and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and beacon placement at the B2M locus and/or the CIITA locus was measured using ddPCR and/or AmpSeq. [0607] FIGs. 10A-10B show beacon placement in iPSC lines ACS-1030 (iPSC1) and ACS- 1026 (iPSC2) at B2M exon 1 and/or CIITA exon 2 for each iPSC line. FIG.10A (left panel) shows beacon placement in iPSC line 1 (ACS-1030) at the B2M locus was between about 15% to about 95% as measured by ddPCR and between about 30% to about 95% as measured by AmpSeq. Additionally, FIG.10A shows beacon placement in iPSC line 1 (ACS-1030) at the CIITA locus was between about 25% to about 45% as measured by ddPCR and between about 30% to about 55% as measured by AmpSeq. FIG.10B shows beacon placement in iPSC line 2 (ACS-1026) at the B2M locus was between about 10% to about 75% as measured by ddPCR. FIG. 10B also shows beacon placement in iPSC line 2 (ACS-1026) at the CIITA locus was between about 10% to about 15% as measured by ddPCR. [0608] Overall, this data showed efficient beacon placement in iPSCs at both the B2M and CIITA loci. 8.3. Example 2: Concurrent beacon placement at B2M and CIITA loci in iPSCs using a dual atgRNA approach at each locus [0609] To demonstrate and optimize concurrent attB beacon placement (i.e., integration recognition site placement or incorporation) at the B2M and CIITA loci within an iPSC, a dual atgRNA approach to beacon placement was used for beacon placement at each locus. The same dual atgRNAs as described in Example 1 were used in this Example. A non-limiting example of a method of generating an iPSC where the method includes concurrent site-specific integration of integration recognition sites in the B2M locus and CIITA locus is shown in FIG.8. [0610] iPSC lines ACS-1030 (iPSC1) and ACS-1026 (iPSC2) were cultured as undifferentiated iPSCs on Cell Basement Membrane (ATCC)-coated plates in Pluripotent Stem Cell SFM XF/FF media (ATCC). Undifferentiated iPSCs were electroporated with prime editor fusion protein (2 micrograms mRNA encoding prime editor fusion protein) and atgRNA (50 picomoles). Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM ROCK-inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, media was changed, and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and beacon placement at the B2M locus and/or the CIITA locus was measured using ddPCR and/or AmpSeq. [0611] FIGs.11A-11B shows concurrent beacon placement at B2M and CIITA loci using the dual atgRNA approach for beacon placement at each locus as described above. FIG.11A shows ddPCR plots, with the top panel showing data for target specific probes and the bottom panel showing data for control probes. FIG. 11B shows a summary histogram from the ddPCR data presented in the FIG.11A. [0612] Overall, FIGs. 11A-11B show successful concurrent beacon placement at B2M and CIITA loci with at least 84.9% beacon placement for B2M and at least 51.3% beacon placement for CIITA. [0613] For iPS cell line generation, electroporated iPSCs were plated on Cell Basement Membrane-coated plates in Pluripotent Stem Cell SFM XF/FF media and allowed to recover. iPSCs were plated at a density suitable to allow the single cells to grow into iPSC colonies that could be picked and expanded for purposes of cell line generation. During initial colony expansion (i.e., prior to picking), iPSCs were grown in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM ROCK inhibitor Y27632. About 7-14 days following plating, colonies were picked, disassociated into clumps (e.g., no less than 4 cells per clump) and re-plated onto a Cell Basement Membrane-coated dish. The picked iPSC lines were expanded, analyzed for beacon placement, and cryopreserved. [0614] Analysis of beacon placement in the iPSC lines generated according to the methods described above is shown in FIGs. 12A-12B. FIG. 12A shows raw ddPCR data for beacon placement at the B2M locus and the CIITA locus for 22 iPSC lines. FIG. 12B is a histogram summarizing ddPCR data for three iPSC lines: line 1, line 8, and line 9. Line 1 and line 9 show 100% bi-allelic beacon placement at both B2M and CIITA loci. AmpSeq was used to confirm the ddPCR data. FIG.13 shows AmpSeq data for 6 iPSC lines including lines 1, 8, 9, 16, 17, and 22. The AmpSeq data did indeed confirm the ddPCR data from FIGs. 12A-12B. Based on the data from FIGs.12A-12B and FIG.13, iPSC lines 16, 17 and 22 showed concurrent, bi-allelic beacon placement at both B2M and CIITA loci. Therefore, these lines were selected for programmable gene insertion (i.e., integration of donor polynucleotide templates) into one or both of the B2M locus and the CIITA locus. 8.4. Example 3: Concurrent Integration of 6 kilobase donor templates at beacons incorporated into the B2M and CIITA loci using electroporation [0615] The iPS cell lines generated in Example 2 were used to assess concurrent integration integrate donor polynucleotide templates at the B2M locus, the CIITA locus, or both. In this Example, a first donor polynucleotide template (pDY-GG-EF1aGFP (SEQ ID NO: 618) about 6 kilobase (kb) in size having an AttP integration recognition site comprising a GG central dinucleotide was targeted to the AttB integration recognition site that was site-specifically integrated at the B2M locus. This Example also included a second donor polynucleotide template (pL1113 (SEQ ID NO: 619) about 5 kb in size having an AttP integration recognition site comprising a GT central dinucleotide that was targeted to the AttB integration recognition site site- specifically integrated at the CIITA locus. [0616] At Day 0, the undifferentiated iPS cell line (line #17) was electroporated with plasmid DNA encoding BxB1 as well as plasmid DNA from the first donor polynucleotide template and/or plasmid DNA from the second donor polynucleotide template. In particular, a single cell suspension of 100,000 iPSCs in 10 µL E1 buffer was combined with plasmid DNA according to the conditions specified in the Table 14.
Figure imgf000242_0001
[0617] Electroporation was performed using a Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM ROCK inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, the media was refreshed without Y27632 and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and beacon placement at the B2M locus and/or the CIITA locus was measured using ddPCR and/or AmpSeq. [0618] FIGs.14A-B illustrates the primer and probe design for detecting integration of donor polynucleotide templates at the B2M locus (FIG. 14A) and the CIITA locus (FIG. 14B) using ddPCR. [0619] FIG.15 shows integration of the first donor polynucleotide template at the B2M locus (site 1) and the second donor polynucleotide template at the CIITA locus (site 2) as determined by ddPCR. In particular, FIG. 15 shows successful integration of the first donor polynucleotide template at the B2M locus (site 1) in both the single and duplex conditions. FIG.15 also illustrates successful integration of the second donor polynucleotide template at the CIITA locus (site 2) in both the single and duplex conditions. In the first “single” condition, integration of the first donor polynucleotide template at the B2M locus (site 1) occurred at about 1% of the B2M locus (see FIG. 15). In the other “single” condition, integration of the second donor polynucleotide template at the CIITA locus (site 2) occurred at about 9% of the CIITA loci (see FIG.15). [0620] In the duplex condition, integration of the first donor polynucleotide templates into the B2M locus (site 1) occurred in about 1.5% of the B2M locus and integration of the second donor polynucleotide template into the CIITA locus occurred at about 14% of the CIITA locus. [0621] Overall, this data showed the ability to concurrently integrate the first and second donor polynucleotides in an iPSC line in a single transduction. 8.5. Example 4: Integration of 31 kilobase adenovirus donor template within beacons incorporated into the CIITA locus in an iPSC [0622] The iPS cell lines generated in Example 2 were used to test integration of a 31 kilobase (kb) donor polynucleotide template at the CIITA locus. In particular, the donor polynucleotide template was a 31kb adenoviral vector (SEQ ID NO: 667) containing an AttP integration recognition site comprising a GG central dinucleotide that was integrated to the AttB integration recognition site site-specifically integrated at exon 2 of the CIITA locus. The adenoviral donor template was introduced into the iPSCs as an adenovirus. [0623] Adenovirus production was performed as described in Ioannidi et al. (bioRxiv 2021.11.01.466786; doi.org/10.1101/2021.11.01.466786) and Wold et al. (Curr. Gene Ther.13, 421–433 (2013)), each of which are herein incorporated by reference in its entireties. [0624] At Day 0, the undifferentiated iPS cell line (line #17) was electroporated with plasmid DNA encoding BxB1. In particular, a single cell suspension of 100,000 iPSCs in 10 µL E1 buffer was combined with 0.5 µg of plasmid DNA encoding BxB1. Electroporation was performed using a Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 0 µM ROCK inhibitor Y27632 and allowed to recover for about 24 hours. [0625] Following the 24-hour recovery period, the electroporated iPSCs were contacted with adenovirus containing the donor polynucleotide template. Various multiplicity of infections (MOIs), including MOIs of 0.01, 0.1, 1, and 10, were used to infect the cells. The different MOIs helped assess dose-dependent impacts on infection and integration. [0626] Following adenovirus infection, iPSCs were cultured for an additional two days prior to harvesting the cells and analyzing for integration of the donor polynucleotide templates. DNA was harvested using a LGC QuickExtract buffer and integration of donor polynucleotide template at the CIITA locus was measured using ddPCR. As noted above, FIG.14B shows a schematic of the primer and probe design for the ddPCR assay used to detect integration of the 31kb donor polynucleotide template at the CIITA locus. [0627] As shown in FIG.16, the 31kb donor polynucleotide template integrated into the CIITA locus in a dose-dependent manner. At an MOI of 10, the 31kb donor polynucleotide integrated into the CIITA locus occurred in about 6% of the CIITA locus. [0628] Overall, this data demonstrates the ability to site-specifically incorporate integration recognition sites into the genome of an iPSC and use these sites along with an integrase to integrate a 31kb donor polynucleotide template at the integration recognition sites. 8.6. Example 5: Beacon placement at CD52 locus in iPSCs having beacons in B2M and CIITA loci [0629] A dual atgRNA approach was used for beacon (also referred to as an integration recognition site) placement at the CD52 locus. A non-limiting example of a method of generating an iPSC where the method includes placing a beacon in the CD52 locus in an iPSC line already having beacons in the B2M and CIITA loci is shown in FIG.8. AtgRNA used in this Example are shown in Table 15. To knockout CD52 expression and place a beacon in the CD52 locus, three forward atgRNAs and four reverse atgRNA all within the 200bp of CD52 exon 1 were designed. After identifying these atgRNAs, forward and reverse atgRNA were combined to make 12 combinations of dual atgRNAs pairs (see Table 15 and FIG.19A).
Figure imgf000244_0001
Figure imgf000245_0001
Figure imgf000246_0001
[0630] Each of these twelve atgRNA pairs were individually electroporated along with a mRNA encoding a gene editor polypeptide (e.g., a Cas9n-RT) into iPSC clone #17 (see FIG.19A for atgRNA pairs). For the electroporation, 3.0 µg of an mRNA encoding the gene editor polypeptide and 0.5 µL (50pmol) of each of the atgRNA RNAF and atgRNA R were added to 100,000 cells of iPS clone #17. Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Clone #17 has perfect beacon placement in both B2M and CIITA loci. After electroporation, genomic DNA was isolated and ddPCR was performed using primers and proved designed to detect beacon placement within the 200bp of CD52 exon 1 (see Table 13). As shown in FIG. 19A, ddPCR analysis revealed the highest performing atgRNA pair, which was pair 6, resulted in 39% beacon placement. Other top performing atgRNA pairs included pairs 2, 5, and 8, each having beacon placement higher than 30% (see FIG.19A). The highest performing pairs (i.e., pairs 2, 5, 6 and 8 (~30% BP)) were used for placing a beacon in the CD52 locus. Cells electroporated with pairs 2, 6, and 8 were subjected to next generation sequencing as an alternative means for detecting beacon placement. As shown in FIG.19B, NGS analysis confirmed that pairs 2, 6, and 8 produced the highest beacon placement, and in fact had higher beacon placement percentages than were detected from ddPCR analysis. [0631] To increase percent beacon placement, atgRNA were designed to target additional regions of CD52. CD52 is a small protein with only two exons, so additional guide atgRNAs are being designed and tested that target exon1-intron 1 junction, intron1-exon2 junction and exon 2. Similar to the atgRNA targeting CD52 exon 1, the atgRNA are designed such that they knockout expression of CD52 while also placing a beacon to be used for programmable gene insertion. In particular, the atgRNA designed to target the intron1-exon2 junction were designed to delete the coding sequence upstream of serine 36. CD52 has 61 amino acids where amino acids 1-24 are the signal peptide and amino acids 37-61 of the propeptide are cleaved . Therefore, as long as placing the beacon removes the first 35 amino acid residues of CD52 it will lose function. [0632] For additional studies, including generating an iPSC line starting with iPSC clone #17 such that the resulting line has beacons in (and knockout of) CD52, B2M, and CIITA, atgRNA pair six was selected to place the beacon in the CD52 locus. In particular, for iPSC line generation, iPSC line #17 was electroporated with an mRNA encoding the gene editor polypeptide and a pair of atgRNA that enable beacon placement (and subsequent knock out of) in the CD52 locus. Electroporated iPSCs were plated on Cell Basement Membrane-coated plates in Pluripotent Stem Cell SFM XF/FF media and allowed to recover. iPSCs were plated at a density suitable to allow the single cells to grow into iPSC colonies that could be picked and expanded for purposes of cell line generation. During initial colony expansion (i.e., prior to picking), iPSCs were grown in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM ROCK inhibitor Y27632. About 7-14 days following plating, colonies were picked, disassociated into clumps (e.g., no less than 4 cells per clump) and re-plated onto a Cell Basement Membrane-coated dish. The picked iPSC lines were expanded, analyzed for beacon placement, and cryopreserved. 8.7. Example 6: Beacon placement at CISH locus in iPSCs having beacons in B2M and CIITA loci [0633] A dual atgRNA approach was used for beacon (also referred to as an integration recognition site) placement at the CISH locus. A non-limiting example of a method of generating an iPSC where the method includes placing a beacon in the CISH locus in an iPSC line already having beacons in each of the B2M and CIITA loci is shown in FIG. 8. AtgRNA used in this Example are shown in Table 16. To knockout CISH and place a beacon in the CISH locus, six pairs of atgRNAs were designed that targeted exon 1 and exon 2 in the CISH locus. After identifying these atgRNAs, forward and reverse atgRNA were combined to make the six atgRNA pairs (see Table 16 and FIGs.20A-20B).
Figure imgf000247_0001
Figure imgf000248_0001
Figure imgf000249_0001
[0634] The six pairs of atgRNA were electroporated along with a mRNA encoding a gene editor polypeptide (e.g., a Cas9n-RT) into iPSC clone #17. For the electroporation, 3.0 µg of an mRNA encoding the gene editor polypeptide and 0.5 µL (50pmol) of each of the atgRNA RNAF and atgRNA R were added to 100,000 cells of iPSC clone #17. Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. As described herein, clone #17 has perfect beacon placement at both the B2M and the CIITA loci. After electroporation, genomic DNA was isolated and ddPCR was performed using primers and probes designed to detect beacon placement at the CISH locus (see Table 13). As shown in FIG. 20A, ddPCR analysis revealed the highest performing atgRNA pair produced about 61% percent beacon placement. gDNA from cells electroporated with the two highest performing atgRNA pairs were kept for NGS analysis to further characterize perfect beacon placement. Cells electroporated in FIG.20A were also subjected to next generation sequencing as an alternative means for detecting beacon placement. FIG. 20B, which shows the NGS analysis, confirmed the same pattern of beacon placement efficiency shown in FIG.20A. [0635] For additional studies, including generating an iPSC line starting with iPSC clone #17 such that the resulting line has beacons in (and knockout of) B2M, CIITA, and CISH, the top performing atgRNA pair (SP01-N01, see FIG. 20A) was electroporated into iPSC line #17. In particular, for iPSC line generation, iPSC line #17 was electroporated with an mRNA encoding the gene editor polypeptide and a pair of atgRNA that enable beacon placement (and subsequent knock out of) in the CISH locus. Electroporated iPSCs were plated on Cell Basement Membrane- coated plates in Pluripotent Stem Cell SFM XF/FF media and allowed to recover. iPSCs were plated at a density suitable to allow the single cells to grow into iPSC colonies that could be picked and expanded for purposes of cell line generation. During initial colony expansion (i.e., prior to picking), iPSCs were grown in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM ROCK inhibitor Y27632. About 7-14 days following plating, colonies were picked, disassociated into clumps (e.g., no less than 4 cells per clump) and re-plated onto a Cell Basement Membrane-coated dish. The picked iPSC lines were expanded, analyzed for beacon placement, and cryopreserved. 8.8. Example 7: Beacon placemen at ADORA2A locus in iPSC having beacons in B2M and CIITA loci [0636] A dual atgRNA approach was used for beacon placement at the ADORA2A locus. A non-limiting example of a method of generating an iPSC where the method includes placing a beacon in the ADORA2A locus in an iPSC line already having beacons in the B2M and CIITA loci is shown in FIG.8. AtgRNA used in this Example are shown in Table 17. To knockout expression of ADORA2A and place a beacon in the ADORA2A locus, five atgRNA pairs were designed that targeted exon 2 of the ADOR2A2 locus. Forward and reverse atgRNA were combined to make the five atgRNA pairs that were tested in this example (see Table 17 and FIGs.21A-21B).
Figure imgf000250_0001
Figure imgf000251_0001
[0637] The five pairs of atgRNA were electroporated along with a mRNA encoding a gene editor polypeptide (e.g., a Cas9n-RT) into iPSC clone #17. For the electroporation, 3.0 µg of an mRNA encoding the gene editor polypeptide and 0.5 µL (50pmol) of each of the atgRNA RNAF and atgRNA R were added to 100,000 cells of iPS clone #17. Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. After electroporation, genomic DNA was isolated and ddPCR was performed using primers and probes designed to detect beacon placement at the ADOR2A2 locus (see Table 13). As shown in FIG. 21A, ddPCR analysis revealed the highest performing atgRNA produced about 58% percent beacon placement. Cells electroporated in FIG. 21A were also subjected to next generation sequencing as an alternative means for detecting beacon placement. FIG.21B, which shows the NGS analysis, confirmed the same pattern of beacon placement efficiency shown in FIG.21A. [0638] For additional studies, including generating an iPSC line starting with iPSC clone #17 such that the resulting line has beacons in (and knockout of) ADORA2A, B2M, and CIITA, the top performing atgRNA pair (SP01-N01, see FIG. 21A) was electroporated into iPSC line #17. In particular, for iPSC line generation, iPSC line #17 was electroporated with an mRNA encoding the gene editor polypeptide and a pair of atgRNA that enable beacon placement (and subsequent knock out of) in the ADORA2A locus. Electroporated iPSCs were plated on Cell Basement Membrane-coated plates in Pluripotent Stem Cell SFM XF/FF media and allowed to recover. iPSCs were plated at a density suitable to allow the single cells to grow into iPSC colonies that could be picked and expanded for purposes of cell line generation. During initial colony expansion (i.e., prior to picking), iPSCs were grown in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM ROCK inhibitor Y27632. About 7-14 days following plating, colonies were picked, disassociated into clumps (e.g., no less than 4 cells per clump) and re-plated onto a Cell Basement Membrane-coated dish. The picked iPSC lines were expanded, analyzed for beacon placement, and cryopreserved. 8.9. Example 8: Concurrent beacon placement at B2M, CIITA, and CD52 loci in iPSCs using a dual atgRNA approach at each locus [0639] To demonstrate and optimize concurrent attB beacon placement (i.e., integration recognition site placement or incorporation) at the B2M, CIITA, and CD52 loci within an iPSC, a dual atgRNA approach to beacon placement was used for beacon placement at each locus. The same dual atgRNAs as described in Example 1 (B2M and CIITA atgRNAs) and Example 5 (CD52 atgRNAs) were used in this Example. A non-limiting example of a method of generating an iPSC where the method includes concurrent site-specific integration of integration recognition sites in the B2M locus, the CIITA locus, and the CD52 locus is shown in FIG.8. [0640] iPSC lines ACS-1030 (iPSC1) and ACS-1026 (iPSC2) were cultured as undifferentiated iPSCs on Cell Basement Membrane (ATCC)-coated plates in Pluripotent Stem Cell SFM XF/FF media (ATCC). Undifferentiated iPSCs were electroporated with 2 µg mRNA encoding a gene editor polypeptide (Cas9n-RT) and 50 picomoles of each atgRNA as described in Table 18.
Figure imgf000252_0001
Figure imgf000253_0001
[0641] To test if we can achieve similar efficiency of triple beacon placement to single beacon placement, we did single and triple beacon placement side by side. For single beacon placement (sample 2 to 6), PE2 mRNA and single pair atgRNA of B2M (B2M bmp001 or bmp003), CIITA (CIITA bmp001 or bmp003) or CD52 mod2 were electroporated into WT iPSC2. For triple (3x) beacon placement (sample 7-9), PE2 mRNA plus B2M bmp001, CIITA bpm001 and CD52 mod2 atgRNA pairs were electroporated into WT iPSC2. For triple (3x) beacon placement (sample 10- 12), PE2 mRNA plus B2M bmp003, CIITA bmp003 and CD52 mod2 atgRNA pairs were electroporated into WT iPSC2 cells. [0642] Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM ROCK-inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, media was changed and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and beacon placement at the B2M locus and/or the CIITA locus was measured using ddPCR. Cells were harvested 72 hours post electroporation and subjected to ddPCR analysis. [0643] FIG. 22 shows ddPCR data of percent beacon placement. The ddPCR data revealed that the beacon placement efficiency is comparable between triple and single beacon placement (see FIG.22). For B2M the efficiency was about 80%, for CIITA the efficiency was between 10- 20%, and for CD52 the efficiency was about 50%. [0644] Overall, this data showed concurrent beacon placement in three different target loci (i.e., B2M, CIITA, and CD52 loci) following a single transfection in wild type iPSC. 8.10. Example 9: Concurrent beacon placement at the TRAC and the AAVS1 loci using a dual atgRNA approach at each locus [0645] To demonstrate and optimize concurrent attB beacon placement (i.e., integration recognition site placement or incorporation) at the TRAC and AAVS1 loci in iPSC clone 17 (i.e., an iPSC clone having beacons already placed at the B2M and CIITA loci), a dual atgRNA approach to beacon placement was used for beacon placement at each locus. AtgRNAs for targeting the TRAC and AAVS1 loci are as described in Table 19. A non-limiting example of a method of generating an iPSC where the method includes concurrent site-specific integration of integration recognition sites in the B2M locus, the CIITA locus, the TRAC locus, and the AAVS1 locus is shown in FIG.8.
Figure imgf000254_0001
[0646] iPSC lines 16, 17, and 29 were cultured as undifferentiated iPSCs on Cell Basement Membrane (ATCC)-coated plates in Pluripotent Stem Cell SFM XF/FF media (ATCC). Undifferentiated iPSCs were electroporated with 2 µg mRNA encoding a gene editor polypeptide (Cas9n-RT) and 50 picomoles of each atgRNA. [0647] Electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM ROCK-inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, media was changed and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and beacon placement at the B2M locus and/or the CIITA locus was measured using ddPCR and/or AmpSeq. [0648] FIG. 23 shows concurrent beacon placement at the TRAC and AAVS1 loci in iPSC clone 17. In particular, FIG. 23 shows ddPCR data of percent beacon placement with the respective pairs with nearly 100% beacon placement at AAVS1, and around 30% beacon placement at TRAC. [0649] Overall, this data demonstrated successful generation of a cell line comprising four beacons placed at four different loci in the genome. 8.11. Example 10: iPSC line generation of iPSC containing beacons in B2M, CIITA, TRAC and AAVS1 loci [0650] To generate iPSC lines having attB beacons (i.e., integration recognition sites) at the TRAC and AAVS1 loci, iPSC line #17, which has a beacon already placed in both the B2M and CIITA loci, was electroporated with dual atgRNAs targeting the TRAC and AAVS1 are described in the preceding examples. A non-limiting example of a method of generating an iPSC cell line where the method includes concurrent site-specific integration of integration recognition sites (beacons) in one or more of the B2M locus, the CIITA locus, the TRAC locus, and the AAVS1 locus is shown in FIG.24. [0651] For these experiments, iPSC clone # 17 was cultured as undifferentiated iPSCs on Cell Basement Membrane (ATCC)-coated plates in Pluripotent Stem Cell SFM XF/FF media (ATCC). Undifferentiated iPSCs were electroporated with 2 µg mRNA encoding a gene editor polypeptide (Cas9n-RT) and atgRNA as described in Examples 2 and 9. In particular, electroporation was performed using the Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM ROCK-inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, media was changed and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and beacon placement at the B2M locus and/or the CIITA locus was measured using ddPCR and/or AmpSeq. As shown in FIG. 24 and FIG. 25, multiple lines were selected and expanded, including iPSC clones 16, 26, 27, 29, 30, and 33. [0652] FIG.25 shows next generation sequencing data for beacon placement at the TRAC and AAVS1 loci in the indicated iPSC lines. Overall, this data demonstrated successful generation of a cells comprising four beacons placed at four different loci in the genome. From this data, iPSC lines containing beacons in each the B2M, CIITA, TRAC, and AAVS1 loci were used for multiplexed programmable gene insertion (i.e., concurrent site-specific integration of four donor polynucleotide templates). 8.12. Example 11: Concurrent site-specific integration of four donor templates in iPS cells containing beacons at B2M, CIITA, TRAC, and AAVS1 loci [0653] iPS cells generated in Example 10 containing beacons at each of the B2M, CIITA, TRAC, and AAVS1 loci were used to assess concurrent integration of donor polynucleotide templates at each of the locus (also referred to as multiplexed programmable gene insertion). Each of the four donor polynucleotide templates included a beacon (i.e., an integration recognition site (e.g., an AttP site)) comprising a different central dinucleotide than the beacons in the other donor polynucleotide templates. Each beacon in each donor polynucleotide template was “paired” with its corresponding (cognate) integration recognition site site-specifically integrated into the iPSC genome at one of the B2M, CIITA, TRAC, and AAVS1 loci (see lines generated in Example 10), such that when contacted with an integration enzyme a recombination event occurs and integration results. [0654] As described in Example 3, a first donor polynucleotide template (pDY-GG-EF1aGFP (SEQ ID NO: 618)) about 6 kilobase (kb) in size and having an AttP integration recognition site comprising a GG central dinucleotide was targeted to the AttB integration recognition site that was site-specifically integrated at the B2M locus. The second donor polynucleotide template (pL1113 (SEQ ID NO: 619)) was about 5 kb in size and included an AttP integration recognition site comprising a GT central dinucleotide that was targeted to the AttB integration recognition site site- specifically integrated at the CIITA locus. The third donor polynucleotide template (pL1220 (SEQ ID NO: 620)) was about 3.8 kb in size and included an AttP integration recognition site comprising a CT central dinucleotide that was targeted to the AttB integration recognition site site-specifically integrated at the TRAC locus. The fourth donor polynucleotide template (pL1218 (SEQ ID NO: 621)) was about 4.0 kb in size and included an AttP integration recognition site comprising an AC central dinucleotide that was targeted to the attB integration recognition site site-specifically integrated at the AAVS1 locus. Sequences of each the donor polynucleotide templates used for multiplexed programmable gene insertion are described in Table 20.
Figure imgf000257_0001
Figure imgf000258_0001
Figure imgf000259_0001
Figure imgf000260_0001
Figure imgf000261_0001
Figure imgf000262_0001
Figure imgf000263_0001
Figure imgf000264_0001
Figure imgf000265_0001
Figure imgf000266_0001
[0655] In a first set of experiments, about 0.8µg of each donor polynucleotide template (about 3.2 µg total) were combined with about 1.0 µg of a plasmid PL193 encoding a BxB1 polypeptide. The final volume was brought to 6 µL and combined with about 100,000 cells of iPSC clones #16 or clone #29 in 10 µL E1 buffer. Similar to clone #17, clones #16 and #29 each have beacons integrated at the B2M, CIITA, TRAC, and AAVS1 loci. The cells-plasmid mixture was electroporated using a Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM ROCK inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, the media was refreshed without Y27632 and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and integration of the donor polynucleotides at each locus was measured using ddPCR and/or AmpSeq. [0656] As shown in FIG.26, concurrent integration of the donor polynucleotide templates at each of the four loci was observed in both iPSC clones #16 and #29. Notably, the efficiency was consistent in both colonies. The highest PGI being at the TRAC locus for both clones, with #29 having only 1% greater PGI than #16. [0657] In a third set of experiments, about 1.0µg of each donor polynucleotide template (about 3.2 µg total) were combined with about 2.0 µg of a mRNA (PL 1325) encoding a BxB1 polypeptide. The final volume was brought to 6 µL and combined with about 100,000 cells of iPSC clones #30 in 10 µL E1 buffer. Similar to clone #17, clone #30 has beacons integrated at the B2M, CIITA, TRAC, and AAVS1 loci. The cells-plasmid mixture was electroporated using a Neon Transfection System with a Neon P10 tip at 1200V, 30 msec, 1 pulse. Following electroporation, the electroporated iPSCs were plated on Cell Basement Membrane coated 48 well plates in Pluripotent Stem Cell SFM XF/FF media supplemented with 10 µM ROCK inhibitor Y27632 and allowed to recover for about 24 hours. After 24 hours, the media was refreshed without Y27632 and cells were cultured for an additional 72 hours. DNA was harvested using LGC QuickExtract buffer and integration of the donor polynucleotides at each locus was measured using ddPCR and/or AmpSeq. As shown in FIG. 27, concurrent integration of the donor polynucleotide templates at each of the four loci was observed in iPSC clone #30. [0658] Overall, this data represents the first successful demonstration of multiplexed PGI at four different loci. 9. SEQUENCE APPENDIX [0659] >NANOGprm-FKBPCasp9-bGHpolyA-ACTB-mFRBFKBPCasp9-CAG-CD47- PDL1-HLAE-3AttB (SEQ ID NO: 616) (corresponding to FIG.3A and the relative positions for each of the features) ATGTATGCTATACGAAGTTATCCGAAGCCGCTAGGTGGTTTGTCTGGTCAACCACCG CGGGCTCAGTGGTGTACGGTACAAACCCAGGAACCCCACTCTAAAAACTTTGTTCCT TTGGAAAACACCTCCCTTCCCCCAGAAACACACACACCCACACGAGATGGGCACGG AGTAGTCTTGAAAGACATGACAAATCACCAGACCTGGGAAGAAGCTAAAGAGCCAG AGGGAAAAAGCCAGAAGTCGACTACCTGGGAGGAGGGATAGACAAGAAACCAAAC TAAAGGAAACTAAGGTAGGTGCTGAAAACAAGTACCATTTTCAACATTAACTGATG CCTTGGCTTCATGCTATAATGCCATGTTGTGTTTCACTATAACCTCAGAGTGAATGA AAGAGGAAAATGGAGCTAGTTGAAATTTCTGCCTAAACTAGCCAGATTTTGAGACA CTAAGTTATCTCAAATCAAGAAATCACCCTAATGAGAATTTCAATAACCTCAGGAAT TTAAGGTGCATGCATCCCCCACCCCCCCCTTTTTTTTTTGAGACGTAGTCCCGCTCTG TTGCCCAGGCTGGAGTACAGTGGCGCGATATCGGCTCACCACAACCTCTGCCTCCCA GGTTCAAGGGATTCTCCCGCCTCAGCTTCCAGAGTAGCTGGGACTACAGACACCCAC CACCATGCGTGGCTAATTTTTGTATTTTTAGTAGAGAGGGGGTTTCGCCATGTTGGC CAGGCTGGTTTCAAACTCCTGACTTCAGGTGATCCGCCTGCCACGGCCTCCCAATTT ACTGGGATTACAGGGGTGGGCCACCGCGCCCGGCCTTTTTCTTAATTTTTAAAAATA TTAAAGTTTTATCCCATTCCTGTTGAACCATATTCCTGATTTAAAAGTTGGAAACGTG GTGAACCTAGAAGTATTTGTTGCTGGGTTTGTCTTCAGGTTCTGTTGCTCGGTTTTCT AGTTCCCCACCTAGTCTGGGTTACTCTGCAGCTACTTTTGCATTACAATGGCCTTGGT GAGACTGGTAGACGGGATTAACTGAGAATTCACAAGGGTGGGTCAGTAGGGGGTGT GCCCGCCAGGAGGGGTGGGTCTAAGGTGATAGAGCCTTCATTATAAATCTAGAGAC TCCAGGATTTTAACGTTCTGCTGGACTGAGCTGGTTGCCTCATGTTATTATGCAGGC AACTCACTTTATCCCAATTTCTTGATGCTTGAGGGAGTGCAGGTGGAAACCATCTCC CCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACAC CGGGATGCTTGAAGATGGAAAGAAAGTTGATTCCTCCCGGGACAGAAACAAGCCCT TTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCC CAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGT GCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAG CTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTCGACGGATTTGGTGATGTCGGT GCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCC TGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGC ACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTG CATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTT GCTGGAGCTGGCGCAGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCT CTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGA TGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCC CAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGA AAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTA ACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTG GACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCC CAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGG ACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGG TCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATT TCCTCCGGAAAAAACTTTTCTTTAAAACATCAGTCGACTAACCTGTGCCTTCTAGTTG CCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACT CCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGT CATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAG AGAATAGCAGGCATGCTGGGGAGTTCCATGTCCTTATATGGACTCATCTTTGCCTAT TGCGACACACACTCAATGAACACCTACTACGCGCTGCAAAGAGCCCCGCAGGCCTG AGGTGCCCCCACCTCACCACTCTTCCTATTTTTGTGTAAAAATCCAGCTTCTTGTCAC CACCTCCAAGGAGGGGGAGGAGGAGGAAGGCAGGTTCCTCTAGGCTGAGCCGAAT GCCCCTCTGTGGTCCCACGCCACTGATCGCTGCATGCCCACCACCTGGGTACACACA GTCTGTGATTCCCGGAGCAGAACGGACCCTGCCCACCCGGTCTTGTGTGCTACTCAG TGGACAGACCCAAGGCAAGAAAGGGTGACAAGGACAGGGTCTTCCCAGGCTGGCTT TGAGTTCCTAGCACCGCCCCGCCCCCAATCCTCTGTGGCACATGGAGTCTTGGTCCC CAGAGTCCCCCAGCGGCCTCCAGATGGTCTGGGAGGGCAGTTCAGCTGTGGCTGCG CATAGCAGACATACAACGGACGGTGGGCCCAGACCCAGGCTGTGTAGACCCAGCCC CCCCGCCCCGCAGTGCCTAGGTCACCCACTAACGCCCCAGGCCTGGTCTTGGCTGGG CGTGACTGTTACCCTCAAAAGCAGGCAGCTCCAGGGTAAAAGGTGCCCTGCCCTGT AGAGCCCACCTTCCTTCCCAGGGCTGCGGCTGGGTAGGTTTGTAGCCTTCATCACGG GCCACCTCCAGCCACTGGACCGCTGGCCCCTGCCCTGTCCTGGGGAGTGTGGTCCTG CGACTTCTAAGTGGCCGCAAGCCACCTGACTCCCCCAACACCACACTCTACCTCTCA AGCCCAGGTCTCTCCCTAGTGACCCACCCAGCACATTTAGCTAGCTGAGCCCCACAG CCAGAGGTCCTCAGGCCCTGCTTTCAGGGCAGTTGCTCTGAAGTCGGCAAGGGGGA GTGACTGCCTGGCCACTCCATGCCCTCCAAGAGCTCCTTCTGCAGGAGCGTACAGAA CCCAGGGCCCTGGCACCCGTGCAGACCCTGGCCCACCCCACCTGGGCGCTCAGTGCC CAAGAGATGTCCACACCTAGGATGTCCCGCGGTGGGTGGGGGGCCCGAGAGACGGG CAGGCCGGGGGCAGGCCTGGCCATGCGGGGCCGAACCGGGCACTGCCCAGCGTGGG GCGCGGGGGCCACGGCGCGCGCCCCCAGCCCCCGGGCCCAGCACCCCAAGGCGGCC AACGCCAAAACTCTCCCTCCTCCTCTTCCTCAATCTCGCTCTCGCTCTTTTTTTTTTTC GCAAAAGGAGGGGAGAGGGGGTAAAAAAATGCTGCACTGTGCGGCGAAGCCGGTG AGTGAGCGGCGCGGGGCCAATCAGCGTGCGCCGTTCCGAAAGTTGCCTTTTATGGCT CGAGCGGCCGCGGCGGCGCCCTATAAAACCCAGCGGCGCGACGCGCCACCACCGCC GAGACCGCGTCCGCCCCGCGAGCACAGAGCCTCGCCTTTGCCGATCCGCCGCCCGTC CACACCCGCCGCCAGATGGCTAGCAGAATCCTGTGGCACGAGATGTGGCACGAGGG CCTGGAGGAGGCTAGCAGACTGTACTTCGGCGAGAGAAACGTGAAAGGAATGTTCG AGGTGCTGGAGCCCCTGCACGCCATGATGGAGAGAGGCCCTCAGACCCTGAAGGAG ACAAGCTTCAACCAAGCCTACGGCAGAGACCTGATGGAGGCCCAAGAGTGGTGCAG AAAGTACATGAAGAGCGGCAACGTGAAGGACCTGCTGCAAGCCTGGGACCTGTACT ACCACGTGTTCAGAAGAATCAGCAAAAGCGGCGGCGGCAGCCGAGGCGTGCAAGT GGAGACCATCAGCCCCGGCGACGGCAGAACCTTCCCCAAGAGAGGGCAGACCTGCG TGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGTTCGACAGCAGCAGAGAC AGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAAGAGGTGATCAGAGGCTGGGA GGAGGGCGTGGCTCAGATGAGCGTGGGGCAGAGAGCCAAGCTGACCATATCCCCCG ATTACGCCTACGGTGCTACGGGCCACCCCGGCATTATCCCCCCCCACGCCACCCTGG TGTTTGACGTGGAGCTGCTGAAACTGGAAAGCGGAGGCGGCTCCGGCGTTGACGGT TTCGGCGATGTGGGCGCCCTGGAGAGCCTGAGAGGCAACGCCGACCTGGCCTACAT CCTGAGCATGGAGCCCTGCGGCCACTGCCTGATCATCAACAACGTGAACTTCTGCAG AGAGAGCGGACTGAGAACGCGTACCGGCAGCAACATCGACTGCGAGAAGCTGAGA AGAAGATTTAGCAGCCTGCATTTTATGGTGGAGGTGAAGGGCGACCTGACCGCCAA GAAGATGGTGCTGGCCCTGCTGGAGCTGGCTCAGCAAGACCACGGCGCCCTGGACT GCTGCGTGGTGGTGATCCTGAGCCACGGCTGCCAAGCCTCGCACCTGCAGTTTCCCG GCGCAGTGTACGGCACCGACGGCTGCCCCGTGAGCGTGGAGAAGATCGTGAACATC TTCAACGGCACAAGCTGCCCTAGCCTGGGCGGCAAGCCCAAATTGTTCTTCATCCAA GCCTGCGGCGGCGAGCAGAAAGACCACGGCTTCGAGGTTGCAAGCACCTCACCGGA AGACGAGTCGCCCGGCAGTAATCCCGAACCCGACGCCACCCCCTTCCAAGAGGGGC TGCGGACATTCGATCAGCTGGACGCCATCAGCAGCCTGCCCACGCCTAGCGACATCT TCGTGAGCTACAGCACCTTCCCCGGCTTCGTGAGCTGGAGAGACCCCAAGAGCGGC AGCTGGTACGTGGAGACCCTGGACGACATCTTCGAGCAGTGGGCCCACAGCGAGGA CCTGCAGAGCCTGCTGCTGAGAGTGGCCAACGCCGTGAGCGTGAAGGGCATCTACA AGCAGATGCCCGGCTGCTTCAACTTCCTGAGAAAGAAACTTTTCTTCAAGACTAGCG TGGACGGCAGCGGCGCCACAAACTTCTCTCTGCTAAAGCAAGCAGGTGATGTTGAA GAAAACCCCGGGCCTATGCCCACGCTACTGCGGGTTTATATAGACGGTCCTCACGGG ATGGGGAAAACCACCACCACGCAACTGCTGGTGGCCCTGGGTTCGCGCGACGATAT CGTCTACGTACCCGAGCCGATGACTTACTGGCAGGTGCTGGGGGCTTCCGAGACAAT CGCGAACATCTACACCACACAACACCGCCTCGACCAGGGTGAGATATCGGCCGGGG ACGCGGCGGTGGTAATGACAAGCGCCCAGATAACAATGGGCATGCCTTATGCCGTG ACCGACGCCGTTCTGGCTCCTCATATCGGGGGGGAGGCTGGGAGCTCACATGCCCC GCCCCCGGCCCTCACCCTCATCTTCGACCGCCATCCCATCGCCGCCCTCCTGTGCTAC CCGGCCGCGCGATACCTTATGGGCAGCATGACCCCCCAGGCCGTGCTGGCGTTCGTG GCCCTCATCCCGCCGACCTTGCCCGGCACAAACATCGTGTTGGGGGCCCTTCCGGAG GACAGACACATCGACCGCCTGGCCAAACGCCAGCGCCCCGGCGAGCGGCTTGACCT GGCTATGCTGGCCGCGATTCGCCGCGTTTACGGGCTGCTTGCCAATACGGTGCGGTA TCTGCAGGGCGGCGGGTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGACGGCCG TGCCGCCCCAGGGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGG GACACGTTATTTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTG TACAACGTGTTTGCCTGGGCCTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCAC GTCTTTATCCTGGATTACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGCTGCAA CTTACCTCCGGGATGGTCCAGACCCACGTCACCACCCCCGGCTCCATACCGACGATC TGCGACCTGGCGCGCACGTTTGCCCGGGAGATGGGGGAGGCTAACTGACTTCTGGG TAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTGGGAATTTTTTGTGTCTCTCAG ATTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTC ATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCT GACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAA CGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCC ACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATG ACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTA CTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCA CGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTT ATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCC AGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGC GGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGC GGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGAGTCGCTGCGCGCTGCC TTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGA CCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATT AGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGG GGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGT GTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTG CGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCG GGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCG GGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAAC CCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGC TCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTG GGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGC GCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGC CTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCG GAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCG GTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCC GCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTC GGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGA GCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGGATGTCTAGATTAGAA GAAGAATTAAGAAGAAGATTAACTGAATGGCCCCTGGTAGCGGCGCTGTTGCTGGG CTCGGCGTGCTGCGGATCAGCTCAGCTACTATTTAATAAAACAAAATCTGTAGAATT CACGTTTTGTAATGACACTGTCGTCATTCCATGCTTTGTTACTAATATGGAGGCACA AAACACTACTGAAGTATACGTAAAGTGGAAATTTAAAGGAAGAGATATTTACACCT TTGATGGAGCTCTAAACAAGTCCACTGTCCCCACTGACTTTAGTAGTGCAAAAATTG AAGTCTCACAATTACTAAAAGGAGATGCCTCTTTGAAGATGGATAAGAGTGATGCT GTCTCACACACAGGAAACTACACTTGTGAAGTAACAGAATTAACCAGAGAAGGTGA AACGATCATCGAGCTAAAATATCGTGTTGTTTCATGGTTTTCTCCAAATGAAAATAT TCTTATTGTTATTTTCCCAATTTTTGCTATACTCCTGTTCTGGGGACAGTTTGGTATTA AAACACTTAAATATAGATCCGGTGGTATGGATGAGAAAACAATTGCTTTACTTGTTG CTGGACTAGTGATCACTGTCATTGTCATTGTTGGAGCCATTCTTTTCGTCCCAGGTGA ATATTCATTAAAGAATGCTACTGGCCTTGGTTTAATTGTGACTTCTACAGGGATATT AATATTACTTCACTACTATGTGTTTAGTACAGCGATTGGATTAACCTCCTTCGTCATT GCCATATTGGTTATTCAGGTGATAGCCTATATCCTCGCTGTGGTTGGACTGAGTCTCT GTATTGCGGCGTGTATACCAATGCATGGCCCTCTTCTGATTTCAGGTTTGAGTATCTT AGCTCTAGCACAATTACTTGGACTAGTTTATATGAAATTTGTGGCTTCCAATCAGAA GACTATACAACCTCCTAGGAAAGCTGTAGAGGAACCCCTTAATGCATTCAAAGAAT CAAAAGGAATGATGAATGATGAAGGCAGCGGCGCCACAAACTTCTCTCTGCTAAAG CAAGCAGGTGATGTTGAAGAAAACCCCGGGCCTATGAGGATATTTGCTGTCTTTATA TTCATGACCTACTGGCATTTGCTGAACGCATTTACTGTCACGGTTCCCAAGGACCTA TATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAA ACAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTAT TCAATTTGTGCATGGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAGACAGA GGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACA GATGTGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGC CGACTACAAGCGAATTACTGTGAAAGTCAATGCCCCATACAACAAAATCAACCAAA GAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGG GCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGT AAGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCAC ACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGA TCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGAACTACCTCTGGCACATCC TCCAAATGAAAGGACTCACTTGGTAATTCTGGGAGCCATCTTATTATGCCTTGGTGT AGCACTGACATTCATCTTCCGTTTAAGAAAAGGGAGAATGATGGATGTGAAAAAAT GTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACG GGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC CGGCCCAATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGC CTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAG AATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATT GAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTT GTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACT GAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGAT AGTTAAGTGGGATCGAGACATGGTAGATGGAACCCTCCTTTTACTCCTCTCGGAGGC CCTGGCCCTTACCCAGACCTGGGCGGGCTCCCACTCCTTGAAGTATTTCCACACTTC CGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCTCTGTGGGCTACGTGGACGA CACCCAGTTCGTGCGCTTCGACAACGACGCCGCGAGTCCGAGGATGGTGCCGCGGG CGCCGTGGATGGAGCAGGAGGGGTCAGAGTATTGGGACCGGGAGACACGGAGCGC CAGGGACACCGCACAGATTTTCCGAGTGAATCTGCGGACGCTGCGCGGCTACTACA ATCAGAGCGAGGCCGGGTCTCACACCCTGCAGTGGATGCATGGCTGCGAGCTGGGG CCCGACGGGCGCTTCCTCCGCGGGTATGAACAGTTCGCCTACGACGGCAAGGATTAT CTCACCCTGAATGAGGACCTGCGCTCCTGGACCGCGGTGGACACGGCGGCTCAGAT CTCCGAGCAAAAGTCAAATGATGCCTCTGAGGCGGAGCACCAGAGAGCCTACCTGG AAGACACATGCGTGGAGTGGCTCCACAAATACCTGGAGAAGGGGAAGGAGACGCT GCTTCACCTGGAGCCCCCAAAGACACACGTGACTCACCACCCCATCTCTGACCATGA GGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTG GCAGCAGGATGGGGAGGGCCATACCCAGGACACGGAGCTCGTGGAGACCAGGCCT GCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGA GCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTACCCGAGCCCGTCACCCTGA GATGGAAGCCGGCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGG TTCTCCTTGGATCTGTGGTCTCTGGAGCTGTGGTTGCTGCTGTGATATGGAGGAAGA AGAGCTCAGGTGGAAAAGGAGGGAGCTACTCTAAGGCTGAGTGGAGCGACAGTGC CCAGGGGTCTGAGTCTCACAGCTTGTAAGCAGTGAAAAAAATGCTTTATTTGTGAAA TTTGTGATGCTATTGCTTTATTGGCCGGCTTGTCGACGACGGCGAACTCCGTCGTCA GGATCATCCGGGGCCGGCTTGTCGACGACGGCGCGCTCCGTCGTCAGGATCATCCG GGGCCGGCTTGTCGACGACGGCGCCCTCCGTCGTCAGGATCATCCGGTGCTATTGCT TTATTTGTGGGCCCG [0660] > PTRE3G-B2M-pA-hPGKprm-Tet3G-pA (2345 bp) (SEQ ID NO: 617) (corresponding to FIG.3B and the relative positions for each of the features) ATGTATGCTATACGAAGTTATCCGAAGCCGCTAGGTGGTTTGTCTGGTCAACCACCG CGGTCTCAGTGGTGTACGGTACAAACCCAGTTTACTCCCTATCAGTGATAGAGAACG TATGAAGAGTTTACTCCCTATCAGTGATAGAGAACGTATGCAGACTTTACTCCCTAT CAGTGATAGAGAACGTATAAGGAGTTTACTCCCTATCAGTGATAGAGAACGTATGA CCAGTTTACTCCCTATCAGTGATAGAGAACGTATCTACAGTTTACTCCCTATCAGTG ATAGAGAACGTATATCCAGTTTACTCCCTATCAGTGATAGAGAACGTATAAGCTTTA GGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCG CCTGGAGCAATTCCACAACACTTTTGTCTTATACCAACTTTCCGTACCACTTCCTACC CTCGTAAAATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGG CCTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGA GAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACAT TGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACT TGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCAC TGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGAT AGTTAAGTGGGATCGAGACATGTAAAACTTGTTTATTGCAGCTTATAATGGTTACAA ATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAG TTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCGGGTTGCGCCTT TTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGG AAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTC ACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCC CTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGC CGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGC GCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGC AGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCC TGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAG TCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGGATGTCTAGACTGGAC AAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCAATGGAGTCGGTATCGAAGG CCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACT GGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGAC AGGCATCATACCCACTCCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGG AACAACGCCAAGTCATACCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTG CATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGC GTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGT GGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAG AGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGAAACAAGCAATT GAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATC ATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGACCGACGCCCT TGACGATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGA TATGCTGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCCGGGTAAAA CTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCAC AAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTA TCTTATCATGTCTGGATC [0661] FKBP-linker-Caspase9 amino acid sequence (SEQ ID NO: 662) MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQE VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSG VDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRR RFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAV YGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSN PEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFE QWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSVD [0662] mFRB-linker-FKBP-Caspase9-P2A-DeltaTK amino acid sequence (SEQ ID NO: 663) MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQA YGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKSGGGSRGVQVETISPGD GRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRG NADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDL TAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVN IFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTF DQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLR VANAVSVKGIYKQMPGCFNFLRKKLFFKTSVDGSGATNFSLLKQAGDVEENPGPMPTL LRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQHRLD QGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDRHPIA ALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERL DLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHI GDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQ LTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN [0663] Alpha tag-CD47-P2A-PDL1-T2A-B2M-HLA-E (SEQ ID NO: 664) MSRLEEELRRRLTEWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNM EAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSD AVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLK YRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVF STAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKF VASNQKTIQPPRKAVEEPLNAFKESKGMMNDEGSGATNFSLLKQAGDVEENPGPMRIF AVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDK NIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGG ADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKT TTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHL VILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEETGSGEGRGSLLT CGDVEENPGPMSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGF HPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQP KIVKWDRDMVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD DTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYN QSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQIS EQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLR CWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTC HVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGS YSKAEWSDSAQGSESHSL [0664] B2M amino acid sequence (SEQ ID NO: 665) MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLL KNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRD M [0665] Tet3G amino acid sequence (SEQ ID NO: 666) MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIE MLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQ LAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLKQAIE LFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLP ADALDDFDLDMLPG [0666] Adenoviral vector 31kb (SEQ ID NO: 667) TGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCG TGGGAACGGGGCGGGTGACGTAGGTTTTAGGGCGGAGTAACTTGTATGTGTTGGGA ATTGTAGTTTTCTTAAAATGGGAAGTGACGTAACGTGGGAAAACGGAAGTGACGAT TTGAGGAAGTTGTGGGTTTTTTGGCTTTCGTTTCTGGGCGTAGGTTCGCGTGCGGTTT TCTGGGTGTTTTTTGTGGACTTTAACCGTTACGTCATTTTTTAGTCCTATATATACTCG CTCTGCACTTGGCCCTTTTTTACACTGTGACTGATTGAGCTGGTGCCGTGTCGAGTGG TGTTTTTTTAATAGGTTTTCTTTTTTACTGGTAAGGCTGACTGTTATGGCTGCCGCTGT GGAAGCGCTGTATGTTGTTCTGGAGCGGGAGGGTGCTATTTTGCCTAGGCAGGAGG GTTTTTCAGGTGTTTATGTGTTTTTCTCTCCTATTAATTTTGTTATACCTCCTATGGGG GCTGTAATGTTGTCTCTACGCCTGCGGGTATGTATTCCCCGGGCTATTTCGGTCGCTT TTTAGCACTGACCGATGTGAATCAACCTGATGTGTTTACCGAGTCTTACATTATGAC TCCGGACATGACCGAGGAGCTGTCGGTGGTGCTTTTTAATCACGGTGACCAGTTTTT TTACGGTCACGCCGGCATGGCCGTAGTCCGTCTTATGCTTATAAGGGTTGTTTTTCCT GTTGTAAGACAGGCTTCTAATGTTTAAATGTTTTTTTGTTATTTTATTTTGTGTTTATG CAGAAACCCGCAGACATGTTTGAGAGAAAAATGGTGTCTTTTTCTGTGGTGGTTCCG GAGCTTACCTGCCTTTATCTGCATGAGCATGACTACGATGTGCTTTCTTTTTTGCGCG AGGCTTTGCCTGATTTTTTGAGCAGCACCTTGCATTTTATATCGCCGCCCATGCAACA AGCTTACATCGGGGCTACGCTGGTTAGCATAGCTCCGAGTATGCGTGTCATAATCAG TGTGGGTTCTTTTGTCATGGTTCCTGGCGGGGAAGTGGCCGCGCTGGTCCGTGCAGA CCTGCACGATTATGTTCAGCTGGCCCTGCGAAGGGACCTACGGGATCGCGGTATTTT TGTTAATGTTCCGCTTTTGAATCTTATACAGGTCTGTGAGGAACCTGAATTTTTGCAA TCATGATTCGCTGCTTGAGGCTGAAGGTGGAGGGCGCTCTGGAGCAGATTTTTACAA TGGCCGGACTTAATATTCGGGATTTGCTTAGAGATATATTGAGAAGGTGGCGAGATG AGAATTATTTGGGCATGGTTGAAGGTGCTGGAATGTTTATAGAGGAGATTCACCCTG AAGGGTTTAGCCTTTACGTCCACTTGGACGTGAGGGCCGTTTGCCTTTTGGAAGCCA TTGTGCAACATCTTACAAATGCCATTATCTGTTCTTTGGCTGTAGAGTTTGACCACGC CACCGGAGGGGAGCGCGTTCACTTAATAGATCTTCATTTTGAGGTTTTGGATAATCT TTTGGAATAAAAAAAAAACATGGTTCTTCCAGCTCTTCCCGCTCCTCCCGTGTGTGA CTCGCAGAACGAATGTGTAGGTTGGCTGGGTGTGGCTTATTCTGCGGTGGTGGATGT TATCAGGGCAGCGGCGCATGAAGGAGTTTACATAGAACCCGAAGCCAGGGGGCGCC TGGATGCTTTGAGAGAGTGGATATACTACAACTACTACACAGAGCGATCTAAGCGG CGAGACCGGAGACGCAGATCTGTTTGTCACGCCCGCACCTGGTTTTGCTTCAGGAAA TATGACTACGTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGGTT GTCTCGGCGCACTCCGTACAGTAGGGATCGTCTACCTCCTTTTGAGACAGAAACCCG CGCTACCATACTGGAGGATCATCCGCTGCTGCCCGAATGTAACACTTTGACAATGCA CAACGTGAGTTACGTGCGAGGTCTTCCCTGCAGTGTGGGATTTACGCTGATTCAGGA ATGGGTTGTTCCCTGGGATATGGTTCTAACGCGGGAGGAGCTTGTAATCCTGAGGAA GTGTATGCACGTGTGCCTGTGTTGTGCCAACATTGATATCATGACGAGCATGATGAT CCATGGTTACGAGTCCTGGGCTCTCCACTGTCATTGTTCCAGTCCCGGTTCCCTGCAG TGTATAGCCGGCGGGCAGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGATGGCGCC ATGTTTAATCAGAGGTTTATATGGTACCGGGAGGTGGTGAATTACAACATGCCAAA AGAGGTAATGTTTATGTCCAGCGTGTTTATGAGGGGTCGCCACTTAATCTACCTGCG CTTGTGGTATGATGGCCACGTGGGTTCTGTGGTCCCCGCCATGAGCTTTGGATACAG CGCCTTGCACTGTGGGATTTTGAACAATATTGTGGTGCTGTGCTGCAGTTACTGTGCT GATTTAAGTGAGATCAGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGCCTTATGCT GCGGGCGGTGCGAATCATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGA CGGAGCGGCGGCGGCAGCAGTTTATTCGCGCGCTGCTGCAGCACCACCGCCCTATCC TGATGCACGATTATGACTCTACCCCCATGTAGGCGTGGACTTCTCCTTCGCCGCCCG TTAAGCAACCGCAAGTTGGACAGCAGCCTGTGGCTCAGCAGCTGGACAGCGACATG AACTTAAGTGAGCTGCCCGGGGAGTTTATTAATATCACTGATGAGCGTTTGGCTCGA CAGGAAACCGTGTGGAATATAACACCTAAGAATATGTCTGTTACCCATGATATGATG CTTTTTAAGGCCAGCCGGGGAGAAAGGACTGTGTACTCTGTGTGTTGGGAGGGAGG TGGCAGGTTGAATACTAGGGTTCTGTGAGTTTGATTAAGGTACGGTGATCTGTATAA GCTATGTGGTGGTGGGGCTATACTACTGAATGAAAAATGACTTGAAATTTTCTGCAA TTGAAAAATAAACACGTTGAAACATAACACAAACGATTCTTTATTCTTGGGCAATGT ATGAAAAAGTGTAAGAGGATGTGGCAAATATTTCATTAATGTAGTTGTGGCCAGAC CAGTCCCATGAAAATGACATAGAGTATGCACTTGGAGTTGTGTCTCCTGTTTCCTGT GTACCGTTTAGTGTAATGGTTAGTGTTACAGGTTTAGTTTTGTCTCCGTTTAAGTAAA CTTGACTGACAATGTTACTTTTGGCAGTTTTACCGTGAGATTTTGGATAAGCTGATA GGTTAGGCATAAATCCAACAGCGTTTGTATAGGCTGTGCCTTCAGTAAGATCTCCAT TTCTAAAGTTCCAATATTCTGGGTCCAGGAAGGAATTGTTTAGTAGCACTCCATTTTC GTCAAATCTTATAATAAGATGAGCACTTTGAACTGTTCCAGATATTGGAGCCAAACT GCCTTTAACAGCCAAAACTGAAACTGTAGCAAGTATTTGACTGCCACATTTTGTTAA GACCAAAGTGAGTTTAGCATCTTTCTCTGCATTTAGTCTACAGTTAGGAGATGGAGC TGGTGTGGTCCACAAAGTTAGCTTATCATTATTTTTGTTTCCTACTGTAATGGCACCT GTGCTGTCAAAACTAAGGCCAGTTCCTAGTTTAGGAACCATAGCCTTGTTTGAATCA AATTCTAGGCCATGGCCAATTTTTGTTTTGAGGGGATTTGTGTTTGGTGCATTAGGTG AACCAAATTCAAGCCCATCTCCTGCATTAATGGCTATGGCTGTAGCGTCAAACATCA ACCCCTTGGCAGTGCTTAGGTTAACCTCAAGCTTTTTGGAATTGTTTGAAGCTGTAA ACAAGTAAAGGCCTTTGTTGTAGTTAATATCCAAGTTGTGGGCTGAGTTTATAAAAA GAGGGCCCTGTCCTAGTCTTAGATTTAGTTGGTTTTGAGCATCAAACGGATAACTAA CATCAAGTATAAGGCGTCTGTTTTGAGAATCAATCCTTAGTCCTCCTGCTACATTAA GTTGCATATTGCCTTGTGAATCAAAACCCAAGGCTCCAGTAACTTTAGTTTGCAAGG AAGTATTATTAATAGTCACACCTGGACCAGTTGCTACGGTCAAAGTGTTTAGGTCGT CTGTTACATGCAAAGGAGCCCCGTACTTTAGTCCTAGTTTTCCATTTTGTGTATAAAT GGGCTCTTTCAAGTCAATGCCCAAGCTACCAGTGGCAGTAGTTAGAGGGGGTGAGG CAGTGATAGTAAGGGTACTGCTATCGGTGGTGGTGAGGGGGCCTGATGTTTGCAGG GCTAGCTTTCCTTCTGACACTGTGAGGGGTCCTTGGGTGGCAATGCTAAGTTTGGAG TCGTGCACGGTTAGCGGGGCCTGTGATTGCATGGTGAGTGTGTTGCCCGCGACCATT AGAGGTGCGGCGGCAGCCACAGTTAGGGCTTCTGAGGTAACTGTGAGGGGTGCAGA TATTTCCAGGTTTATGTTTGACTTGGTTTTTTTGAGAGGTGGGCTCACAGTGGTTACA TTTTGGGAGGTAAGGTTGCCGGCCTCGTCCAGAGAGAGGCCGTTGCCCATTTTGAGC GCAAGCATGCCATTGGAGGTAACTAGAGGTTCGGATAGGCGCAAAGAGAGTACCCC AGGGGGACTCTCTTGAAACCCATTGGGGGATACAAAGGGAGGAGTAAGAAAAGGC ACAGTTGGAGGACCGGTTTCCGTGTCATATGGATACACGGGGTTGAAGGTATCTTCA GACGGTCTTGCGCGCTTCATCTGCAACAACATGAAGATAGTGGGTGCGGATGGACA GGAACAGGAGGAAACTGACATTCCATTTAGATTGTGGAGAAAGTTTGCAGCCAGGA GGAAGCTGCAATACCAGAGCTGGGAGGAGGGCAAGGAGGTGCTGCTGAATAAACT GGACAGAAATTTGCTAACTGATTTTAAGTAAGTGATGCTTTATTATTTTTTTTTATTA GTTAAAGGGAATAAGATCCCCGGGTACTCTAGTTAATCGACTTAGGCGACCTGACAT TAACTAGAGGATCTTGATGTAATCCAGGGTTAGGACAGTTGCAAATCACAGTGAGA ACACAGGGTCCCCTGTCCCGCTCAACTAGCAGGGGGCGCTGGGTAAACTCCCGAAT CAGGCTACGGGCAAGCTCTCCCTGGGCGGTAAGCCGGACGCCGTGCGCCGGGCCCT CGATATGATCCTCGGGCAATTCAAAGTAGCAAAACTCACCGGAGTCGCGGGCAAAG CACTTGTGGCGGCGACAGTGGACCAGGTGTTTCAGGCGCAGTTGCTCTGCCTCTCCA CTTAACATTCAGTCGTAGCCGTCCGCCGAGTCCTTTACCGCGTCAAAGTTAGGAATA AATTGATCCGGATAGTGGCCGGGAGGTCCCGAGAAGGGGTTAAAGTAGACCGATGG CACAAACTCCTCAATAAATTGCAGAGTTCCAATGCCTCCAGAGCGCGGCTCAGAGG ACGAGGTCTGCAGAGTTAGGATTGCCTGACGAGGCGTGAATGAAGAGCGGCCGGCG CCGCCGATCTGAAATGTCCCGTCCGGACGGAGACCAAGCGAGGAGCTCACCGACTC GTCGTTGAGCTGAATACCTCGCCCTCTGATTGTCAGGTGAGTTATACCCTGCCCGGG CGACCGCACCCTGTGACGAAAGCCGCCCGCAAGCTGCGCCCCTGAGTTAGTCATCTG AACTTCGGCCTGGGCGTCTCTGGGAAGTACCACAGTGGTGGGAGCGGGACTTTCCTG GTACACCAGGGCAGCGGGCCAACTACGGGGATTAAGGTTATTACGAGGTGTGGTGG TAATAGCCGCCTGTTCCAGGAGAATTCGGTTTCGGTGGGCGCGTATTCCGTTGACCC GGGATATCATGTGGGGTCCCGCGCTCATGTAGTTTATTCGGGTTGAGTAGTCTTGGG CAGCTCCAGCCGCAAGTCCCATTTGTGGCTGGTAACTCCACATGTAGGGCGTGGGAA TTTCCTTGCTCATAATGGCGCTGACAACAGGTGCTGGCGCCGGGTGTGGCCGCTGGA GATGACGTAGTTTTCGCGCTTAAATTTGAGAAAGGGCGCGAAACTAGTCCTTAAGA GTCAGCGCGCAGTATTTACTGAAGAGAGCCTCCGCGTCTTCCAGCGTGCGCCGAAGC TGATCTTCGCTTTTGTGATACAGGCAGCTGCGGGTGAGGGATCGCAGAGACCTGTTT TTTATTTTCAGCTCTTGTTCTTGGCCCCTGCTCTGTTGAAATATAGCATACAGAGTGG GAAAAATCCTGTTTCTAAGCTCGCGGGTCGATACGGGTTCGTTGGGCGCCAGACGCA GCGCTCCTCCTCCTGCTGCTGCCGCCGCTGTGGATTTCTTGGGCTTTGTCAGAGTCTT GCTATCCGGTCGCCTTTGCTTCTGTGTGGCCGCTGCTGTTGCTGCCGCTGCCGCCGGT GCAGTATGGGCTGTAGAGATGACGGTAGTAATGCAGGATGTTACGGGGGAAGGCCA CGCCGTGATGGTAGAGAAGAAAGCGGCGGGCGAAGGAGATGTTGCCCCCACAGTCT TGCAAGCAAGCAACTATGGCGTTCTTGTGCCCGCGCCATGAGCGGTAGCCTTGGCGC TGTTGTTGCTCTTGGGCTAACGGCGGCGGCTGCTTGGACTTACCGGCCCTGGTTCCA GTGGTGTCCCATCTACGGTTGGGTCGGCGAACGGGCAGTGCCGGCGGCGCCTGAGG AGCGGAGGTTGTAGCCATGCTGGAACCGGTTGCCGATTTCTGGGGCGCCGGCGAGG GGAATGCGACCGAGGGTGACGGTGTTTCGTCTGACACCTCTTCGACCTCGGAAGCTT CCTCGTCTAGGCTCTCCCAGTCTTCCATCATGTCCTCCTCCTCCTCGTCCAAAACCTC CTCTGCCTGACTGTCCCAGTATTCCTCCTCGTCCGTGGGTGGCGGCGGCAGCTGCAG CTTCTTTTTGGGTGCCATCCTGGGAAGCAAGGGCCCGCGGCTGCTGCTGATAGGGCT GCGGCGGCGGGGGGATTGGGTTGAGCTCCTCGCCGGACTGGGGGTCCAAGTAAACC CCCCGTCCCTTTCGTAGCAGAAACTCTTGGCGGGCTTTGTTGATGGCTTGCAATTGG CCAAGAATGTGGCCCTGGGTAATGACGCAGGCGGTAAGCTCCGCATTAGGCGGGCG GGATTGGTCTTCGTAGAACCTAATCTCGTGGGCGTGGTAGTCCTCAGGTACAAATTT GCGAAGGTAAGCCGACGTCCACAGCCCCGGAGTGAGTTTCAACCCCGGAGCCGCGG ACTTTTCGTCAGGCGAGGGACCCTGCAGCTCAAAGGTACCGATAATTTGACTTTCGT TAAGCAGCTGCGAATTGCAAACCAGGGAGCGGTGCGGGGTGCATAGGTTGCAGCGA CAGTGACACTCCAGTAGACCGTCACCGCTCACGTCTTCCATTATGTCAGAGTGGTAG GCAAGGTAGTTGGCTAGCTGCAGAAGGTAGCAGTGGCCCCAAAGCGGCGGAGGGC ATTCGCGGTACTTAATGGGCACAAAGTCGCTAGGAAGTGCACAGCAGGTGGCGGGC AAGATTCCTGAGCGCTCTAGGATAAAGTTCCTAAAGTTCTGCAACATGCTTTGACTG GTGAAGTCTGGCAGACCCTGTTGCAGGGTTTTAAGCAGGCGTTCGGGGAAAATGAT GTCCGCCAGGTGCGCGGCCACGGAGCGCTCGTTGAAGGCCGTCCATAGGTCCTTCA AGTTTTGCTTTAGCAGTTTCTGCAGCTCCTTGAGGTTGCACTCCTCCAAGCACTGCTG CCAAACGCCCATGGCCGTCTGCCAGGTGTAGCATAGAAATAAGTAAACGCAGTCGC GGACGTAGTCGCGGCGCGCCTCGCCCTTGAGCGTGGAATGAAGCACGTTTTGCCCA AGGCGGTTTTCGTGCAAAATTCCAAGGTAGGAGACCAGGTTGCAGAGCTCCACGTT GGAGATCTTGCAGGCCTGGCGTACGTAGCCCTGTCGAAAGGTGTAGTGCAATGTTTC CTCTAGCTTGCGCTGCATCTCCGGGTCAGCAAAGAACCGCTGCATGCACTCAAGCTC CACGGTAACGAGCACTGCGGCCATCATTAGTTTGCGTCGCTCCTCCAAGTCGGCAGG CTCGCGCGTTTGAAGCCAGCGCGCTAGCTGCTCGTCGCCAACTGCGGGTAGGCCCTC CTCTGTTTGTTCTTGCAAATTTGCATCCCTCTCCAGGGGCTGCGCACGGCGCACGATC AGCTCACTCATGACTGTGCTCATGACCTTGGGGGGTAGGTTAAGTGCCGGGTAGGCA AAGTGGGTGACCTCGATGCTGCGTTTTAGTACGGCTAGGCGCGCGTTGTCACCCTCG AGTTCCACCAACACTCCAGAGTGACTTTCATTTTCGCTGTTTTCCTGTTGCAGAGCGT TTGCCGCGCGCTTCTCGTCGCGTCCAAGACCCTCAAAGATTTTTGGCACTTCGTTGA GCGAGGCGATATCAGGTATGACAGCGCCCTGCCGCAAGGCCAGCTGCTTGTCCGCT CGGCTGCGGTTGGCACGGCAGGATAGGGGTATCTTGCAGTTTTGGAAAAAGATGTG ATAGGTGGCAAGCACCTCTGGCACGGCAAATACGGGGTAGAAGTTGAGGCGCGGGT TGGGCTCGCATGTGCCGTTTTCTTGGCGTTTGGGGGGTACGCGCGGTGAGAATAGGT GGCGTTCGTAGGCAAGGCTGACATCCGCTATGGCGAGGGGCACATCGCTGCGCTCTT GCAACGCGTCGCAGATAATGGCGCACTGGCGCTGCAGATGCTTCAACAGCACGTCG TCTCCCACATCTAGGTAGTCGCCATGCCTTTCGTCCCCCCGCCCGACTTGTTCCTCGT TTGCCTCTGCGTTGTCCTGGTCTTGCTTTTTATCCTCTGTTGGTACTGAGCGGTCCTCG TCGTCTTCGCTTACAAAACCTGGGTCCTGCTCGATAATCACTTCCTCCTCCTCAAGCG GGGGTGCCTCGACGGGGAAGGTGGTAGGCGCGTTGGCGGCATCGGTGGAGGCGGTG GTGGCGAACTCAGAGGGGGCGGTTAGGCTGTCCTTCTTCTCGACTGACTCCATGATC TTTTTCTGCCTATAGGAGAAGGAAATGGCCAGTCGGGAAGAGGAGCAGCGCGAAAC CACCCCCGAGCGCGGACGCGGTGCGGCGCGACGTCCCCCAACCATGGAGGACGTGT CGTCCCCGTCCCCGTCGCCGCCGCCTCCCCGGGCGCCCCCAAAAAAGCGGATGAGG CGGCGTATCGAGTCCGAGGACGAGGAAGACTCATCACAAGACGCGCTGGTGCCGCG CACACCCAGCCCGCGGCCATCGACCTCGGCGGCGGATTTGGCCATTGCGCCCAAGA AGAAAAAGAAGCGCCCTTCTCCCAAGCCCGAGCGCCCGCCATCACCAGAGGTAATC GTGGACAGCGAGGAAGAAAGAGAAGATGTGGCGCTACAAATGGTGGGTTTCAGCA ACCCACCGGTGCTAATCAAGCATGGCAAAGGAGGTAAGCGCACAGTGCGGCGGCTG AATGAAGACGACCCAGTGGCGCGTGGTATGCGGACGCAAGAGGAAGAGGAAGAGC CCAGCGAAGCGGAAAGTGAAATTACGGTGATGAACCCGCTGAGTGTGCCGATCGTG TCTGCGTGGGAGAAGGGCATGGAGGCTGCGCGCGCGCTGATGGACAAGTACCACGT GGATAACGATCTAAAGGCGAACTTCAAACTACTGCCTGACCAAGTGGAAGCTCTGG CGGCCGTATGCAAGACCTGGCTGAACGAGGAGCACCGCGGGTTGCAGCTGACCTTC ACCAGCAACAAGACCTTTGTGACGATGATGGGGCGATTCCTGCAGGCGTACCTGCA GTCGTTTGCAGAGGTGACCTACAAGCATCACGAGCCCACGGGCTGCGCGTTGTGGCT GCACCGCTGCGCTGAGATCGAAGGCGAGCTTAAGTGTCTACACGGAAGCATTATGA TAAATAAGGAGCACGTGATTGAAATGGATGTGACGAGCGAAAACGGGCAGCGCGC GCTGAAGGAGCAGTCTAGCAAGGCCAAGATCGTGAAGAACCGGTGGGGCCGAAAT GTGGTGCAGATCTCCAACACCGACGCAAGGTGCTGCGTGCACGACGCGGCCTGTCC GGCCAATCAGTTTTCCGGCAAGTCTTGCGGCATGTTCTTCTCTGAAGGCGCAAAGGC TCAGGTGGCTTTTAAGCAGATCAAGGCTTTTATGCAGGCGCTGTATCCTAACGCCCA GACCGGGCACGGTCACCTTTTGATGCCACTACGGTGCGAGTGCAACTCAAAGCCTG GGCACGCGCCCTTTTTGGGAAGGCAGCTACCAAAGTTGACTCCGTTCGCCCTGAGCA ACGCGGAGGACCTGGACGCGGATCTGATCTCCGACAAGAGCGTGCTGGCCAGCGTG CACCACCCGGCGCTGATAGTGTTCCAGTGCTGCAACCCTGTGTATCGCAACTCGCGC GCGCAGGGCGGAGGCCCCAACTGCGACTTCAAGATATCGGCGCCCGACCTGCTAAA CGCGTTGGTGATGGTGCGCAGCCTGTGGAGTGAAAACTTCACCGAGCTGCCGCGGA TGGTTGTGCCTGAGTTTAAGTGGAGCACTAAACACCAGTATCGCAACGTGTCCCTGC CAGTGGCGCATAGCGATGCGCGGCAGAACCCCTTTGATTTTTAAACGGCGCAGACG GCAAGGGTGGGGGTAAATAATCACCCGAGAGTGTACAAATAAAAGCATTTGCCTTT ATTGAAAGTGTCTCTAGTACATTATTTTTACATGTTTTTCAAGTGACAAAAAGAAGT GGCGCTCCTAATCTGCGCACTGTGGCTGCGGAAGTAGGGCGAGTGGCGCTCCAGGA AGCTGTAGAGCTGTTCCTGGTTGCGACGCAGGGTGGGCTGTACCTGGGGACTGTTGA GCATGGAGTTGGGTACCCCGGTAATAAGGTTCATGGTGGGGTTGTGATCCATGGGA GTTTGGGGCCAGTTGGCAAAGGCGTGGAGAAACATGCAGCAGAATAGTCCACAGGC GGCCGAGTTGGGCCCCTGTACGCTTTGGGTGGACTTTTCCAGCGTTATACAGCGGTC GGGGGAAGAAGCAATGGCGCTACGGCGCAGGAGTGACTCGTACTCAAACTGGTAAA CCTGCTTGAGTCGCTGGTCAGAAAAGCCAAAGGGCTCAAAGAGGTAGCATGTTTTT GAGTGCGGGTTCCAGGCAAAGGCCATCCAGTGTACGCCCCCAGTCTCGCGACCGGC CGTATTGACTATGGCGCAGGCGAGCTTGTGTGGAGAAACAAAGCCTGGAAAGCGCT TGTCATAGGTGCCCAAAAAATATGGCCCACAACCAAGATCTTTGACAATGGCTTTCA GTTCCTGCTCACTGGAGCCCATGGCGGCAGCTGTTGTTGATGTTGCTTGCTTCTTTAT GTTGTGGCGTTGCCGGCCGAGAAGGGCGTGCGCAGGTACACGGTTTCGATGACGCC GCGGTGCGGCTGGTGCACACGGACCACGTCAAAGACTTCAAACAAAACATAAAGAA GGGTGGGCTCGTCCATGGGATCCACCTCAAAAGTCATGTCTAGCGCGTGGGCGGAG TTGGCGTAGAGAAGGTTTTGGCCCAGGTCTGTGAGTGCGCCCATGGACATAAAGTTA CTGGAGAATGGGATGCGCCAAAGGGTGCGATCGCAAAGAAACTTTTTCTGGGTAAT GCTGTCAACTGCGGTCTTGCCTATAAGCGGATAGGGGAAGTTAGCAGGGTAGGCCT GTCCTTCGCGCATGGTGGGGGCAAGGTAGCCAACAAATCCAGAGTTGTTGTGTTGGT GTAGGATGCCCACCTGTTGGTAGTCCTTGTATTTAGTATCATCCACCACCTGACGGC TCATGGGCTGGAAGTTTCTAAAGAAGGAGTACATGCGGTCCTTGTAGCTCTCTGGGA TATAGAAGCCCTGGTAGCCAATGTTATAGTTAGCTAGCATTTGTACCAGGAACCAGT CTTTGGTCATGTTACACTGGGCAACGTTGTAACCCTCCCCGTCAACTGAGCGCTTAA TTTCAAACTCGTTGGGGGTAAGCAGGCGGTCATTGCCAGGCCAGCTGACAGAAGAG TCAAAGGTAATGGCCACCTTCTTAAAGGTGTGGTTGAGGTAAAAGGTTCCATCTAGG TAGGGTATAGAGCCAGAGTAGGTGTAATAAGGGTCGTAGCCCGAGCCCAGTGATGG GGTTTCCTTAGTCTTAAGGCGCGTGAAGGCCCAGCCGCGGAAAGCCGCCCAGTTGC GGGAGGGGATGGATATGGGCACGTTGGTAGCGTTGGCGGGTATAGGGTAGAGCATG TTGGCGGCGGAGAGATAGTCGTTAAAGGACTGGTCGTTGGTGTCGTTTCTAAGCATG GCCTCAAGCGTGGAGGCGGTGTTGTGGGCCATGGGGAAGAAGGTGGCGTAAAGGCA AATGCTATCAAACTTAATGCTGGCTCCGTCAACCCTTAGGTCATTTCCTAGGGAGCT CTGCAGAACCATGTTAACATCCTTCCTGAAGTTCCACTCGTAGGTGTATGAGCCCGG CAGGAGAAGGAGGTTTTTAATGGCAAAGAACTTCTGAGGCACCTGGATGTGGAAGG GCACATAGCGACCATTGCCCAGCAACATTGAGCGGTAGCGCAGGCCAGCATTGCGG TGGTGGTTAAATGGGTTGACGTTGTCCATATAGTCAAGGGACCAGCGTGCTCCAAGG TTAATGTAGCAGTCCACTAGCCCGGGAGCCACCACTCGCTTGTTCATGTAGTCGTAG GTGTTTGGGTTATCAGAAATTTTTACGTTGGAAGGACTGTACTTTAGCTTGTCGGGC AAATACAGCGCTATGTTGGAGTACAGGAAATTTCTCCACAGGTTGGCATTTAGATTG ATTTCCATGGCAAAATTATTTCCAACTCTTATTTCATTTTTATCTGAAAATTCTGTAG CATCTTTTTCCCATCCATTTTCCTGACCTGTTTTAGGTTTTACCTTGGTAAGAGTCTCT GTATTAATCACACCTCCCAGTGGAAAGCAGTAATTTGGAAGTTCATCTTCAGTTCCA TGATTTTCAATAATTCTAACATCTGGATCATAGCTGTCAACAGCCTGATTCCACATA GAAAAGTACCTGGTTCTATCACCAATGGAATCAAGCAAAAGCTGGTATGAAAGCTC TGTGTTTCTGTCTTGCAAATCTACAACAGCATTCAACTGCGATGCTTGGCCCGCCAG AACACCCATATTACCCGTGCTGTTGTAATACATTAGACCAATAAAATTGTCCCTAAA AGCAATGTAATTAGGCCTGTTGGGCATAGATTGTTGGCCCATTAGTTCTCGTGAGTT ACCTTCCTTAATAGTGGGCATGTAAGAAATATGAGTGTCTGGGGTTTCTATATCTAC ATCTTCACTGTACAATACCACTTTAGGAGTCAAGTTATCACCATTGCCTGCGGCTGC CTCAGTAGTTGAGAAAAATTGCATTTCCACTTGACTTTCTAGCTTTCCATTTTGTTGC TTTACAAGAATGCCTTGCCCTCCATTTTCATTTGTGGGTTTTGCATATGAACCGTAAC ATGGTTTCATTGGGGTAGTCTTTTTTAGGACTCTCCCAGCTGCATGATTAATTTCTGT TTCGTACCACTGAGATTCTCCTATTTGAGGTTCAGGTTGAAATGTTTTATCGGCATAT TTAGGTGTTTGACCTTCGACACCTATTTGAATACCCTCCTTTGTAATATTTATACCAG AATAAGGCGCCTGCCCAAATACGTGAGTTTTTTGCTGCTCAGCTTGCTCGTCTACTTC GTCTTCGTTGTCATCGTCCTCTTCTTCTAGGTTTATTTCAAGAGCAGTAGCAGCTTCA TCCCATTCGCAAGGATTTGGGGCACCCTTGGGAGCCAGGGCGTTGTAGGCAGTGCC AGAGTAGGGCTTAAAAGTAGGGCCCCTGTCCAGCACGCCGCGGATGTCAAAGTACG TGGAAGCCATGTCCAGCACACGGTTATCACCCACAGCTAGGGTGAACCGCGCCTTGT ACGAGTACGCAGTATCCTCACGGTCCACAGGGATGAACCGCAGCGTCAAACGCTGG GACCGGTCTGTGGTCACGTCGTGCGTAGGCGCCACCGTGGGGTTTCTAAACTTGTTA TTCAGGCTGAAGTACGTCTCGGTGGCGCGGGCAAACTGCACCAGCCCGGGGCTCAG GTACTCCGAGGCGTCCTGGCCCGAGATGTGCATGTAAGACCACTGCGGCATCATCG AAGGGGTAGCCATCTTGGAAAGCGGGCGCGCGGCGGCTCAGCAGCTCCTCTGGCGG CGACATGGACGCATACATGACACACATACGACACGTTAGCTATCAGAAGCATCGTC GGCGCTTCAGGGATTGCACCCCCAGACCCACGATGCTGTTCAGTGTGCTTTGCCAGT TGCCACTGGCTACGGGCCGCAACGATCGCGGACCGCTGGCGGCGCGGCGCAGGGAC GCGCGGCTAGGACGGGTTACAACAACGGCGGTCGGGCCTGGCAGCACAGGTTTCTG CTGGGTGTCGGCGGGGGGAGGCAGGTCCAGCGTTACGGGTGTGTGCTGGCCCAGCA CTCCGGTAGCCATGGGCGCGATGGGACGGGTGGTGGGCAGGCCTTGCTTTAGTGCCT CCTCGTACGAGGGAGGCTCGTCTATTTGCGTCACCAGAGTTTCTTCCCTGTCGGGGC GCGGACGCTTTTCGCCACGCCCCTCTGGAGACACTGTCTCCACGGCCGGTGGAGGCT CCTCTACGGGAGGGCGGGGATCAAGCTTACTGTTAATCTTATTTTGCACTGCCTGGT TGGCCAGGTCCACCACCCCGCTAATGCCAGAGGCCAGGCCATCTACCACCTTTTGTT GGAAATTTTGCTCTTTCAACTTATCCCTCAGCATCTGGCCTGTGCTGCTGTTCCAGGC CTTGCTGCCATAGTTCTTAACGGTGGAACCGAAATTTTTAATGCCGCTCCACAGCGA GCCCCAGCTGAAGGCGCCACCGCTCATATTGCTGGTGCCGATATCTTGCCAGTTTCC CATGAACGGGCGCGAGCCGTGTCGCGGGGCCAGAGACGCAAAGTTGATGTCTTCCA TTCTACAAAATAGTTACAGGACCAAGCGAGCGTGAGAGTCCAGACTTTTTATTTTGA TTTTTCCACATGCAACTTGTTTTTAATCAGTGTCTCTGCGCCTGCAAGGCCACGGATG CAATTCCGGGCACGGCGCCAATCGCCGCGGCGATCAGTGGAATAAGGAGGGGCAGG ATACCGCCGCGCATGCGACGGTGCGACGCGCGCCGCCGCCGGTGGTGCGCACGACG CATGCCGCCCGTCAGGCCGTGGCCGGCCATGCCCCTCCTACGGTGCATTCTTCCTCG GAATCCCGGCACCGGGAAACGGAGGCGGCAGGTGAGGGCCATATCTGCAAGAACC ACAAAGACCGGCTTTTAAACGATGCTGGGGTGGTAGCGCGCTGTTGGCAGCACCAG GGTCCTGCCTCCTTCGCGAGCCACCCTGCGCACGGAAATCGGGGCCAGCACGGGCT GGCGACGGCGACGGCGGCGGCGGGTTCCAGTGGTGGTTCGGCGTCGGGTAGTTGCT CGTCTTCTGGGGCGGTAGGTGTAGCCACGATAGCCGGGGGTAGGCGCAATGGAAGG ATGTAGGGCATATTCGGGCAGTAGCGCGCTGGCGGCGCCGTACTTCCTCGAACGGC GCGGGCGCCGGGGGGCTGAAACGCGAAACATCCACGGGTCCGTTTGCACCTCCGTA GAGGTCTTGGACGCGGCCGCAGCGACCGCCTGCACCGCGGCATCCGCCACCGCTGA GGCAACCGGGGACGTTTGTGTCTCCATGCCCTCTGTGGCGGTGGCAATACTGGTGCT ACTGGTAGTGGGTATCTGAACGTCCACGGTCTGCACGCCCAGTCCCGGCGCCACCTG CTTGATTGGCCGCACGCGGACCTCGGGCTCCAGCCCAGGTTCCACGGTCATTTTTTC CAAGACATCTTCCAGTCGCTGGCGCTTGGGTACCATCAGCTGCACGGTGGGTGCCAA GTCACCAGACTCGCGCTTTAGGCCGCGCTTTTCTTCGGACGGTGCAAGCGCGGGCAG CACCTGCTGCAGTGTTACGGGCTTTAGGCTAGGTGTTGGGTTGCCCTCGTCCAGCGG CAACGCCAGCATGTCCTTATGCCGCTTTCCGTAGGCAAACTCCCCGAGGCGCTCGTT GGCCTGCTCAAGCAGGTCCTCGTCGCCGTACACCTCATCATACACGCGCTTGTAGGT GCGGGTGGAGCGCTCACCGGGCGTAAAGACTACGGTGGTGCCGGGTCGCAAAACAC GTTTTACGCGTCGACCTTTCCACTGTACCCGTCGCCTGGGCGCGGTAGCGTGCAGCA GTTCCACCTCGTCGTCAAGTTCATCATCATCATCTTTCTTTTTCTTTTTGACCCGCTTT AGCTTTCGGGGCTTGTAATCCTGCTCTTCCTTCTTCGGGGGGCCATAGATCTCCGGCG CGATGACCTGGAGCATCTCTTCTTTGATTTTGCGCTTGGACATAGCTTCGTTGCGCGC CGCCGCCGCTGGATACATACAACAGTACGAGTCTAAGTAGTTTTTTCTTGCAATCTA GTTGCGCGGGGGGCGGGTGCGCACGGGCACGCGCAGGCCGCTAACCGAGTCGCGCA CCCAATACACGTTGCCCCTGCGACCCTGAGTCATAGCACTAATGGCCGCGGCTGCTG CGGCGGCCGCTCGTCGCCTGGACCTGGGGGGCACAGTGACAATACCCGCGGCCAGC CTTCGAGCGGCCCGCATGGCCGCCCGTCGGCCGGTGCGACGTGCGCGGTTAAGCAG GGCCGCCGCCGCGCGTTGGGCGGCAGTGCCGGGTCGGCGGCGGTGGCGACGTGCTA CGCGCCTCCGCCGTCTCTTCATTTTAGCATAGCGCCGGGCTCCGCGCACCACGGTCT GAATGGCCGCGTCCACTGTGGACACTGGTGGCGGCGTGGGCGTGTAGTTGCGCGCC TCCTCCACCACCGCGTCGATGGCGTCATCGACGGTGGTGCGCCCAGTGCGGCCGCGT TTGTGCGCGCCCCAGGGCGCGCGGTAGTGCCCGCGCACGCGCACTGGGTGTTGGTC GGAGCGCTTCTTGGCCCCGCCAAACATCTTGCTTGGGAAGCGCAGGCCCCAGCCTGT GTTATTGCTGGGCGATATAAGGATGGACATGCTTGCTCAAAAAGTGCGGCTCGATA GGACGCGCGGCGAGACTATGCCCAGGGCCTTGTAAACGTAGGGGCAGGTGCGGCGT CTGGCGTCAGTAATGGTCACTCGCTGGACTCCTCCGATGCTGTTGCGCAGCGGTAGC GTCCCGTGATCTGTGAGAGCAGGAACGTTTTCACTGACGGTGGTGATGGTGGGGGCT GGCGGGCGCGCCAAAATCTGGTTCTCGGGAAAGCGATTGAACACGTGGGTCAGAGA GGTAAACTGGCGGATGAGTTGGGAGTAGACGGCCTGGTCGTTGTAGAAGCTCTTGG AGTGCACGGGCAACAGCTCGGCGCCCACCACCGGAAAGTTGCTGATCTGGCGCGTG GAGCGGAAGGTCACGGGGTCTTGCATCATGTCTGGCAACGACCAGTAGACCTGCTC CGAGCCGCAGGTTACGTCAGGAGTGCAAAGCAGGGTCCATGAGCGGATTCCGGTCT GAGGGTCGCCGTAGTTGTATGCAAGGTACCAGCTGCGGTACTGGGTGAAGGTGCTG TCATTGCTTATTAGGTTGTAACTGCGTTTCTTGCTGTCCTCTGTCAGGGGTTTGATCA CCGGTTTCTTCTGAGGCTTCTCGACCTCGGGTTGCGCAGCGGGGGCGGCAGCTTCGG CCGCTGCTTCGGCCTCAGCGCGCTTCTCCTCAGCCCGTGTGGCAAAGGTGTCGCCGC GAATGGCATGATCGTTCATGTCCTCCACCGGCTGCATTGCCGCGGCTGCCGCGTTGG AGTTCTCTTCCGCGCCGCTGCCACTGCTGTTGCTGCCGCCTGCGCCACCCCCGCCCTG TTCGGTGTCATCTTTCAAGCTCGCCTGGTAGGCGTCCACATCCAACAGTGCGGGAAT GTTACCACCCTCCAGATCATCGTAGGTGATCCTAAAGCCCTCCTGGAAGGGTTGCCG CTTGCGGATGCCCAACAAGTTGCTCAGGCGGCTGTGGGTGAAGTCCACCCCGCATCC TGGCAGCAAAATGATGTCTGGATGGAAGGCTTCGTTTGTATATACCCCAGGCATGAC AAGACCAGTGACGGGGTCAAACCCCAGTCTGAAGTTGCGGGTGTCAAACTTTACCC CGATGTCGCTTTCCAGAACCCCGTTCTGTCTGCCCACTTTCAAGTAGTGCTCCACGAT CGCGTTGTTCATAAGGTCTATGGTCATGGTCTCGGAGTAGTTGCCCTCGGGCAGCGT GAACTCCACCCACTCGTATTTCAGCTCCACCTGATTGTCCTTAGTAGGCAAGCGCGA CACCATCACCCGCGCCTTAAACTTATTGGTAAACATGAACTCGTTCACATTTGGCAT GTTGGTATGCAGGATGGTTTTCAGGTCGCCGCCCCAGTGCGACCGGTCGTCAAGATT GATGGTCTGTGTGCTTGCCTCCCCCGGGCTGTAGTCATTGTTTTGAATGACCGTGGTC AGAAAGTTGCTGTGGTCGTTCTGGTAGTTCAGGGATGCCACATCCGTTGACTTGTTG TCCACCAGGTACACACGGGTGGTGTCGAATAGGGGTGCCAACTCAGAGTAACGGAT GCTGTTTCTCCCCCCGGTAGGCCGCAGGTACCGCGGAGGCACAAACGGCGGGTCCA GGGGAGCATCGAAGGGAGAACCCAGCGCCGCCGCCACTGGCGCCGCGCTCACCACA CTCTCGTAGGAGGGAGGAGGACCTTCCTCATACATCGCCGCGCGCCGCATACTAAG GGGAATACAAGAAAACCAACGCTCGGTGCCATGGCCTTGGTGAGTTTTTTATTTTGC ATCATGCTTTTTTTTTTTTAAAACATTCTCCCCAGCCTGGGGCGAAGGTGCGCAAAC GGGTTGCCACTCCCTCCCAAATCCAGGACGCTGCTGTCGTCTGCCGAGTCATCGTCC TCCCACACCAGACCCCGCTGACGGTCGTGCCTTTGACGACGGGTGGGCGGGCGCGG GCCTGGCACGTCCCTGTGCTCCTGCGCGTACGTCTTCCATCTACTCATCTTGTCCACT AGGCTCTCTATCCCGTTGTTGGGAAATGCCGGAGGCAGGTTTTTTCGCGCTGCGGCT GCAGCAGCGAGTTGTTTAGGTACTCCTCCTCGCCCAGCAGGCGCGGGCGGGTGGTG CGAGTGCTGGTAAGAGACCCTATCAAGCTTGGAAATGGGCTACTAGCATCTGACCG CGGGGCCGCAGCGCCTAGATCGGACAAGCTGCTTGGCCTGCGGAAGCTTTCCTTTCG CAGCGCCGCCTCTGCCTGCTCGCGCTGTTGCAACTCTAGCAGGGTCTGCGGTTGCGG GGAAAACACGCTGTCGTCTATGTCGTCCCAGAGGAATCCATCGTTACCCTCGGGCAC CTCGAATCCCCCGGTGTAGAAACCAGGGGGCGGTAGCCAGTGCGGGTTCAAGATGG CATTGGTGAAATACTCGGGGTTCACGGCGGCCGCGCGATGCAAGTAGTCCATTAGG CGGTTGATAAACGGCCGGTTTGAGGCATACATGCCCGGTTCCATGTTGCGCGCGGTC ATGTCCAGCGCCACGCTGGGCGTTACCCCGTCGCGCATCAGGTTAAGGCTCACGCTC TGCTGCACGTAGCGCAAAATGCGCTCCTCCTCGCTGTTTAAACTGTGCAACGAGGGG ATCTTCTGCCGCCGGTTGGTCAGCAGGTAGTTTAGGGTTGCCTCCAGGCTGCCCGTG TCCTCCTGCCCCAGCGCGCGGCTGACACTTGTAATCTCCTGGAAAGTATGCTCGTCC ACATGCGCCTGACCTATGGCCTCGCGGTACAGTGTCAGCAAGTGACCTAGGTATGTG TCCCGGGACACGCTGCCACTGTCCGTGAAGGGCGCTATTAGCAGCAGCAACAGGCG CGAGTTGGGCGTCAGCAAGCTAGACACGGTCGCGCGGTCGCCTGTGGGAGCCCGCA CCCCCCACAGCCCCTGCAAGTTTTTGAAAGCCTGGCTCAGGTTTACGGTCTGCAGGC CTTGTCTACTGGTCTGGAAAAAATAGTCTGGCCCAGACTGGTACACCTCACTTTGCG GTGTCTCAGTCACCATTAGCCGCAGTGCGCTCACAAAGTTGGTGTAGTCCTCCTGTC CCCGCGGCACGTTGGCGGGCTGTGTACTCAGGAAGGCGTTTAGTGCAACCATGGAG CCCAGGTTGCCCTGCTGCTGCGCGCGCTCACGCTGCGCCACGGCCTCGCGCACATCC CCCACCAGCCGGTCCAGGTTGGTCTGCACGTTGCCGCTGTTGTAACGAGCCACGCGC TGAAGCAGCGCGTCGTAGACCAGGCCGGCCTCGTCGGGCCGGATGGCCCTGTTTTCG GCCAGCGCGTTTACGATCGCCAGCACCTTCTCGTGCGTGGGGTTTGCGCGCGCCGGG ACCACCGCTTCCAGAATTGCGGAGAGCCGGTTGGCCTGCGGCTGCTGCCGGAACGC GTCAGGATTGCGCGCAGTCAGCGACATGATGCGGTCCATGACCTGGCGCCAGTCGT CCGTGGAGTTAAGGCCGGACGGCTGGCTCTGCAGCGCCGCCCGCACCGCCGGGTCC GTTGCGTCTTGCATCATCTGATCAGAAACATCACCGCTTAGTACTCGCCGTCCTCTG GCTCGTACTCATCGTCCTCGTCATATTCCTCCACGCCGCCGACGTTGCCAGCGCGCG CGGGTGCCACCGCCAGCCCAGGTCCGGCCCCAGCTGCCTCCAGGGCGCGTCGGCTT GGGGCCCAGCGCAGGTCAGCGCCCGCGTCAAAGTAGGACTCGGCCTCTCTATCGCC GCTGCCCGTGCCAGCCAGGGCCCTTTGCAGGCTGTGCATCAGCTCGCGGTCGCTGAG CTCGCGCCGCCGGCTCACGCTCACGGCCTTGTGGATGCGCTCGTTGCGATAAACGCC CAGGTCGTCGCTCAAGGTAAGCACCTTCAGCGCCATGCGCATGTAGAACCCCTCGAT CTTTACCTCCTTGTCTATGGGAACGTAAGGGGTATGGTATATCTTGCGGGCGTAAAA CTTGCCCAGGCTAAGCATGGAATAGTTGATGGCGGCCACCTTGTCAGCCAGGCTCAA GCTGCGCTCCTGCACCACTATGCTCTGCAGGATGTTTATCAAATCGAGCAGCCAGCG GCCCTCGGGCTCTACTATGTTTAGCAGCGCATCCCTGAATGCCTCGTTGTCCCTGCTG TGCTGCACTATAAGGAACAGCTGCGCCATGAGCGGCTTGCTATTTGGGTTTTGCTCC AGCGCGCTTACAAAGTCCCACAGATGCATCAGTCCTATAGCCACCTCCTCGCGCGCC ACAAGCGTACGCACGTGGTTGTTAAAGCTTTTTTGAAAGTTAATCTCCTGGTTCACC GTCTGCTCGTATGCGGTTACCAGGTCGGCGGCCGCCACGTGTGCGCGCGCGGGACTA ATCCCGGTTCGCGCGTCGGGCTCAAAGTCCTCCTCGCGCAGCAACCGCTCGCGATTC AGGCCATGCCGCAGCTCGCGCCCTGCGTGGAACTTTCGATCCCGCATCTCCTCGGGC TCCTCTCCCTCGCGGTCGCGAAACAGGTTCTGCCGCGGCACGTACGCCTCACGCGTA TCACGCTTCAGCTGCACCCTTGGGTGCCGCTCAGGAGAGGGCGCTCCTAGCCGCGCC AGGCCCTCGCCCTCCTCCAAGTCCAGGTAGTGCCGGGCCCGGCGCCGCGGGGGTTC GTAATCACCATCTGCTGCCGCGTCAACCGCGGATGTCGCCCCTCCTGACGCGGTAGG AGGAGGGGAGGGTGCCCTGCATGTCTGCCGCTGCTCTTGCTCTTGCCGCTGCTGAGG AGGGGGGCGCATCTGCCGCAGCACCGGATGCATCTGGGAAAAGCAAAAAAGGGGC TCGTCCCTGTTTCCGGAGGAATTTGCAAGCGGGGTCTTGCATGACGGGGAGGCAAA CCCCCGTTCGCCGCAGTCCGGCCGGTCCGAGACTCGAACCGGGGGTCCCGCGACTC AACCCTTGGAAAATAACCCTCCGGCTACAGGGAGCGAGCCACTTAATGCTTTCGCTT TCCAGCCTAACCGCTTACGCTGCGCGCGGCCAGTGGCCAAAAAAGCTAGCGCAGCA GCCGCCGCGCCTGGAAGGAAGCCAAAAGGAGCACTCCCCCGTTGTCTGACGTCGCA CACCTGGGTTCGACACGCGGGCGGTAACCGCATGGATCACGGCGGACGGCCGGATA CGGGGCTCGAACCCCGGTCGTCCGCCATGATACCCTTGCGAATTTATCCACCAGACC ACGGAAGAGTGCCCGCTTACAGGCTCTCCTTTTGCACGCTAGAGCGTCAACGATTGC GCGCGCCTGACCGGCCAGAGCGTCCCGACCATGGAGCACTTTTTGCCGCTGCGCAAC ATCTGGAACCGCGTCCGCGACTTTCCGCGCGCCTCCACCACCGCCGCCGGCATCACC TGGATGTCCAGGTACATCTACGGATATCATCGCCTTATGTTGGAAGATCTCGCCCCC GGAGCCCCGGCCACCCTACGCTGGCCCCTCTACCGCCAGCCGCCGCCGCACTTTTTG GTGGGATACCAGTACCTGGTGCGGACTTGCAACGACTACGTATTTGACTCGAGGGCT TACTCGCGTCTCAGGTACACCGAGCTCTCGCAGCCGGGTCACCAGACCGTTAACTGG TCCGTTATGGCCAACTGCACTTACACCATCAACACGGGCGCATACCACCGCTTTGTG GACATGGATGACTTCCAGTCTACCCTCACGCAGGTGCAGCAGGCCATATTAGCCGA GCGCGTTGTCGCCGACCTAGCCCTGCTTCAGCCGATGAGGGGCTTCGGGGTCACACG CATGGGAGGAAGAGGGCGCCACCTACGGCCAAACTCCGCCGCCGCCGCAGCGATAG ATGCAAGAGATGCAGGACAAGAGGAAGGAGAAGAAGAAGTGCCGGTAGAAAGGCT CATGCAAGACTACTACAAAGACCTGCGCCGATGTCAAAACGAAGCCTGGGGCATGG CCGACCGCCTGCGCATTCAGCAGGCCGGACCCAAGGACATGGTGCTTCTGTCGACC ATCCGCCGTCTCAAGACCGCCTACTTTAATTACATCATCAGCAGCACCTCCGCCAGA AACAACCCCGACCGCCGCCCGCTGCCGCCCGCCACGGTGCTCAGCCTACCTTGCGAC TGTGACTGGTTAGACGCCTTTCTCGAGAGGTTTTCCGATCCGGTCGATGCGGACTCG CTCAGGTCCCTCGGCGGCGGAGTACCTACACAACAATTGTTGAGATGCATCGTTAGC GCCGTATCCCTGCCGCATGGCAGCCCCCCGCCAACCCATAACCGGGACATGACGGG CGGCGTCTTCCAACTGCGCCCCCGCGAGAACGGCCGCGCCGTCACCGAGACCATGC GCCGTCGCCGCGGGGAGATGATCGAGCGCTTTGTCGACCGCCTCCCGGTGCGCCGTC GTCGCCGCCGTGTCCCCCCTCCCCCACCGCCGCCAGAAGAAGAAGAAGGGGAGGCC CTTATGGAAGAGGAGATTGAAGAAGAAGAAGAGGCCCCTGTAGCCTTTGAGCGCGA GGTGCGCGACACTGTCGCCGAGCTCATCCGTCTTCTGGAGGAGGAGTTAACCGTGTC GGCGCGCAACTCCCAGTTTTTCAACTTCGCCGTGGACTTCTACGAGGCCATGGAGCG CCTTGAGGCCTTGGGGGATATCAACGAATCCACGTTGCGACGCTGGGTTATGTACTT CTTCGTGGCAGAACACACCGCCACCACCCTCAACTACCTCTTTCAGCGCCTGCGAAA CTACGCCGTCTTCGCCCGGCACGTGGAGCTCAATCTCGCGCAGGTGGTCATGCGCGC CCGCGATGCCGAAGGGGGCGTGGTCTACAGCCGCGTCTGGAACGAGGGAGGCCTCA ACGCCTTCTCGCAGCTCATGGCCCGCATTTCCAACGACCTCGCCGCCACCGTGGAGC GAGCCGGACGCGGAGATCTCCAGGAGGAAGAGATCGAGCAGTTCATGGCCGAGATC GCCTATCAAGACAACTCAGGAGACGTGCAGGAGATTTTGCGCCAGGCCGCCGTCAA CGACACCGAAATTGATTCTGTCGAACTCTCTTTCAGGTTCAAGCTCACCGGGCCCGT CGTCTTCACGCAGAGGCGCCAGATTCAGGAGATCAACCGCCGCGTCGTCGCGTTCGC CAGCAACCTACGCGCGCAGCACCAGCTCCTGCCCGCGCGCGGCGCCGACGTGCCCC TGCCCCCTCTCCCGGCGGGTCCGGAGCCCCCCTACCTCCGGGGGCTCGCCCGCGTCA CCGCTTTTAGATGCATCATCCAAGGACACCCCCGCGGCCCACCGCCCGCCGCGCGGT ACCGTAGTCGCGCCGCGGGGATGCGGCCTCTTGCAAGCCATCGACGCCGCCACCAA CCAGCCCCTGGAAATTAGGTATCACCTGGATCTAGCCCGCGCCCTGACCCGTCTATG CGAGGTAAACCTGCAGGAGCTCCCGCCTGACCTGACGCCGCGGGAGCTCCAGACCA TGGACAGCTCCCATCTGCGCGATGTTGTCATCAAGCTCCGACCGCCGCGCGCGGACA TCTGGACTTTGGGCTCGCGCGGCGTGGTGGTCCGATCCACCGTAACTCCCCTCGAGC AGCCAGACGGTCAAGGACAAGCAGCCGAAGTAGAAGACCACCAGCCAAACCCGCC AGGCGAGGGGCTCAAATTCCCACTCTGCTTCCTTGTGCGCGGTCGTCAGGTCAACCT CGTGCAGGATGTACAGCCCGTGCACCGCTGCCAGTACTGCGCACGTTTTTACAAAAG CCAGCACGAGTGTTCGGCCCGTCGCAGGGACTTCTACTTTCACCACATCAATAGCCA CTCCTCCAATTGGTGGCGGGAGATCCAGTTCTTCCCGATCGGCTCGCATCCTCGCAC CGAGCGTCTCTTTGTCACCTACGATGTAGAGACCTATACTTGGATGGGGGCCTTTGG GAAGCAGCTCGTGCCCTTCATGCTGGTCATGAAGTTCGGCGGAGATGAGCCTCTAGT GACTGCCGCGCGAGACCTAGCCGCGAACCTTGGATGGGACCGCTGGGAACAAGACC CGCTTACCTTCTACTGCATCACCCCAGAAAAAATGGCCATAGGTCGCCAGTTTAGGA CCTTTCGCGACCACCTGCAAATGCTAATGGCCCGTGACCTGTGGAGCTCATTCGTCG CTTCCAACCCTCATCTTGCAGACTGGGCCCTTTCAGAGCACGGGCTCAGCTCCCCTG AAGAGCTCACCTACGAGGAACTTAAAAAATTGCCTTCCATCAAGGGCATCCCGCGC TTCTTGGAACTTTACATTGTGGGCCACAACATCAACGGCTTTGACGAGATCGTGCTC GCCGCCCAGGTAATTAACAACCGTTCCGAGGTGCCGGGACCCTTCCGCATCACACGC AACTTTATGCCTCGCGCGGGAAAGATACTCTTCAACGATGTCACCTTCGCCCTGCCA AATCCGCGTTCCAAAAAGCGCACGGACTTTTTGCTCTGGGAGCAGGGCGGATGCGA CGACACTGACTTCAAATACCAGTACCTCAAAGTCATGGTCAGGGACACCTTTGCGCT CACCCACACCTCGCTCCGGAAGGCCGCGCAGGCATACGCGCTACCCGTAGAAAAGG GATGCTGCGCCTACCAGGCCGTCAACCAGTTCTACATGCTAGGCTCTTACCGTTCGG AGGCCGACGGGTTTCCGATCCAAGAGTACTGGAAAGACCGCGAAGAGTTTGTCCTC AACCGCGAGCTGTGGAAAAAAAGGGACAGGATAAGTATGACATCATCAAGGAAAC CCTGGACTACTGCGCCCTAGACGTGCAGGTCACCGCCGAGCTGGTCAACAAGCTGC GCGACTCCTACGCCTCCTTCGTGCGTGACGCGGTAGGTCTCACAGACGCCAGCTTCA ACGTCTTCCAGCGTCCAACCATATCATCCAACTCACATGCCATCTTCAGGCAGATAG TCTTCCGAGCAGAGCAGCCCGCCCGTAGCAACCTCGGTCCCGACCTCCTCGCTCCCT CGCACGAACTATACGATTACGTGCGCGCCAGCATCCGCGGTGGAAGATGCTACCCT ACATATCTTGGAATACTCAGAGAGCCCCTCTACGTTTACGACATTTGCGGCATGTAC GCCTCCGCGCTCACCCACCCCATGCCATGGGGTCCCCCACTCAACCCATACGAGCGC GCGCTTGCCGCCCGCGCATGGCAGCAGGCGCTAGACTTGCAAGGATGCAAGATAGA CTACTTCGACGCGCGCCTGCTGCCCGGGGTCTTTACCGTGGACGCAGACCCCCCGGA CGAGACGCAGCTAGACCCCCTACCGCCATTCTGCTCGCGCAAGGGCGGCCGCCTCTG CTGGACCAACGAGCGCCTACGCGGAGAGGTAGCCACCAGCGTTGACCTTGTCACCC TGCACAACCGCGGTTGGCGCGTGCACCTGGTGCCCGACGAGCGCACCACCGTCTTTC CCGAATGGCGGTGCGTTGCGCGCGAATACGTGCAGCTAAACATCGCGGCCAAGGAG CGCGCCGATCGCGACAAAAACCAAACCCTGCGCTCCATCGCCAAGTTGCTGTCCAA CGCCCTCTACGGGTCGTTTGCCACCAAGCTTGACAACAAAAAGATTGTCTTTTCTGA CCAGATGGATGCGGCCACCCTCAAAGGCATCACCGCGGGCCAGGTGAATATCAAAT CCTCCTCGTTTTTGGAAACTGACAATCTTAGCGCAGAAGTCATGCCCGCTTTTCAGA GGGAGTACTCACCCCAACAGCTGGCCCTCGCAGACAGCGATGCGGAAGAGAGTGAG GACGAACGCGCCCCCACCCCCTTTTATAGCCCCCCTTCAGGAACACCCGGTCACGTG GCCTACACCTACAAACCAATCACCTTCCTTGATGCCGAAGAGGGCGACATGTGTCTT CACACCCTGGAGCGAGTGGACCCCCTAGTGGACAACGACCGCTACCCCTCCCACTTA GCCTCCTTCGTGCTGGCCTGGACGCGAGCCTTTGTCTCAGAGTGGTCCGAGTTTCTAT ACGAGGAGGACCGCGGAACACCGCTCGAGGACAGGCCTCTCAAGTCTGTATACGGG GACACGGACAGCCTTTTCGTCACCGAGCGTGGACACCGGCTCATGGAAACCAGAGG TAAGAAACGCATCAAAAAGCATGGGGGAAACCTGGTTTTTGACCCCGAACGGCCAG AGCTCACCTGGCTCGTGGAATGCGAGACCGTCTGCGGGGCCTGCGGCGCGGATGCC TACTCCCCGGAATCGGTATTTCTCGCGCCCAAGCTCTACGCCCTCAAAAGTCTGCAC TGCCCCTCGTGCGGCGCCTCCTCCAAGGGCAAGCTGCGCGCCAAGGGCCACGCCGC GGAGGGGCTGGACTATGACACCATGGTCAAATGCTACCTGGCCGACGCGCAGGGCG AAGACCGGCAGCGCTTCAGCACCAGCAGGACCAGCCTCAAGCGCACCCTGGCCAGC GCGCAGCCCGGAGCGCACCCCTTCACCGTGACCCAGACTACGCTGACGAGGACCCT GCGCCCGTGGAAAGACATGACCCTGGCCCGTCTGGACGAGCACCGACTACTGCCGT ACAGCGAAAGCCGCCCCAACCCGCGAAACGAGGAGATATGCTGGATCGAGATGCCG TAGAGCAGGTGACCGAGCTGTGGGACCGCCTGGAACTGCTTGGTCAAACGCTCAAA AGCATGCCTACGGCGGACGGTCTCAAACCGTTGAAAAACTTTGCTTCCTTGCAAGAA CTGCTATCGCTGGGCGGCGAGCGCCTTCTGGCGGATTTGGTCAGGGAAAACATGCG AGTCAGGGACATGCTTAACGAAGTGGCCCCCCTGCTCAGGGATGACGGCAGCTGCA GCTCTCTTAACTACCAGTTGCAGCCGGTAATAGGTGTGATTTACGGGCCCACCGGCT GCGGTAAGTCGCAGCTGCTCAGGAACCTGCTTTCTTCCCAGCTGATCTCCCCTACCC CGGAAACCGTTTTCTTCATCGCCCCGCAGGTAGACATGATCCCCCCATCTGAACTCA AAGCGTGGGAAATGCAAATCTGTGAGGGTAACTACGCCCCTGGGCCGGATGGAACC ATTATACCGCAGTCTGGCACCCTCCGCCCGCGCTTTGTAAAAATGGCCTATGACGAT CTCATCCTGGAACACAACTATGACGTTAGTGATCCCAGAAATATCTTCGCCCAGGCC GCCGCCCGTGGGCCCATTGCCATCATTATGGACGAATGCATGGAAAATCTTGGAGGT CACAAGGGCGTCTCCAAGTTCTTCCACGCATTTCCTTCTAAGCTACATGACAAATTT CCCAAGTGCACCGGATACACTGTGCTGGTGGTTCTGCACAACATGAATCCCCGGAG GGATATGGCTGGGAACATAGCCAACCTAAAAATACAGTCCAAGATGCATCTCATAT CCCCACGTATGCACCCATCCCAGCTTAACCGCTTTGTAAACACTTACACCAAGGGCC TGCCCCTGGCAATCAGCTTGCTACTGAAAGACATTTTTAGGCACCACGCCCAGCGCT CCTGCTACGACTGGATCATCTACAACACCACCCCGCAGCATGAAGCTCTGCAGTGGT GCTACCTCCACCCCAGAGACGGGCTTATGCCCATGTATCTGAACATCCAGAGTCACC TTTACCACGTCCTGGAAAAAATACACAGGACCCTCAACGACCGAGACCGCTGGTCC CGGGCCTACCGCGCGCGCAAAACCCCTAAATAAAGACAGCAAGACACTTGCTTGAT CCAAATCCAAACAGAGTCTGGTTTTTTATTTATGTTTTAAACCGCATTGGGAGGGGA GGAAGCCTTCAGGGCAGAAACCTGCTGGCGCAGATCCAACAGCTGCTGAGAAACGA CATTAAGTTCCCGGGTCAAAGAATCCAATTGTGCCAAAAGAGCCGTCAACTTGTCAT CGCGGGCGGATGAACGGGAAGCTGCACTGCTTGCAAGCGGGCTCAGGAAAGCAAA GTCAGTCACAATCCCGCGGGCGGTGGCTGCAGCGGCTGAAGCGGCGGCGGAGGCTG CAGTCTCCAACGGCGTTCCAGACACGGTCTCGTAGGTCAAGGTAGTAGAGTTTGCGG GCAGGACGGGGCGACCATCAATGCTGGAGCCCATCACATTCTGACGCACCCCGGCC CATGGGGGCATGCGCGTTGTCAAATATGAGCTCACAATGCTTCCATCAAACGAGTTG GTGCTCATGGCGGCGGCGGCTGCTGCAAAACAGATACAAAACTACATAAGACCCCC ACCTTATATATTCTTTCCCACCCTTAACCACGCCCAGATCCTCTAGAAAGCTTCTCGA GGATATCGCGGCCGCGTCGACGGTACCCGGGCCCACAAATAAAGCAATAGCATCAC AAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTC ATCGAGCTCGAGATCTGGCGAAGGCGATGGGGGTCTTGAAGGCGTGCTGGTACTCC ACGATGCCCAGCTCGGTGTTGCTGTGCAGCTCCTCCACGCGGCGGAAGGCGAACAT GGGGCCCCCGTTCTGCAGGATGCTGGGGTGGATGGCGCTCTTGAAGTGCATGTGGCT GTCCACCACGAAGCTGTAGTAGCCGCCGTCGCGCAGGCTGAAGGTGCGGGCGAAGC TGCCCACCAGCACGTTATCGCCCATGGGGTGCAGGTGCTCCACGGTGGCGTTGCTGC GGATGATCTTGTCGGTGAAGATCACGCTGTCCTCGGGGAAGCCGGTGCCCACCACCT TGAAGTCGCCGATCACGCGGCCGGCCTCGTAGCGGTAGCTGAAGCTCACGTGCAGC ACGCCGCCGTCCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATG GCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCC GTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAGGGCGCCTTTGGTGC TCTTCATCTTGTTGGTCATGCGGCCCTGCTCGGGGGTGCCCTCTCCGCCGCCCACCAG CTCGAACTCCACGCCGTTCAGGGTGCCGGTGATGCGGCACTCGATCTTCATGGCGGG CATGGTGGCGACCGGTAGCTGGGTTTGTACCGTACACCACTGAGACCGCGGTGGTTG ACCAGACAAACCACCGCTAGCGGCTTCGGATAACTTCGTTAACTTGTTTATTGCAGC TTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTT TTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAACGCGATCAA GCTAGCTTGCTAGACTCGACTGACTATAATAATAAAACGCCAACTTTGACCCGGAAC GCGGAAAACACCTGAGAAAAACACCTGGGCGAGTCTCCACGTAAACGGTCAAAGTC CCCGCGGCCCTAGACAAATATTACGCGCTATGAGTAACACAAAATTATTCAGATTTC ACTTCCTCTTATTCAGTTTTCCCGCGAAAATGGCCAAATCTTACTCGGTTACGCCCAA ATTTACTACAACATCCGCCTAAAACCGCGCGAAAATTGTCACTTCCTGTGTACACCG GCGCACACCAAAAACGTCACTTTTGCCACATCCGTCGCTTACATGTGTTCCGCCACA CTTGCAACATCACACTTCCGCCACACTACTACGTCACCCGCCCCGTTCCCACGCCCC GCGCCACGTCACAAACTCCACCCCCTCATTATCATATTGGCTTCAATCCAA
10. EQUIVALENTS AND INCORPORATION BY REFERENCE [0667] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and every individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. §1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. [0668] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it is understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS: 1. A method of generating a pleiopluripotent cell, the method comprising: (a) site-specifically incorporating at least a first integration recognition site into a genome of a pleiopluripotent cell.
2. The method of claim 1, wherein site-specifically incorporating the at least first integration recognition site is effected by introducing into the pleiopluripotent cell: (i) a gene editor protein or a polynucleotide encoding a gene editor protein or a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein; (ii) at least a first pair of guide RNAs, wherein the first paired guide RNAs have domains that are capable of guiding the gene editor protein or prime editor fusion protein to nick the pleiopluripotent cell genome at sites respectively flanking the specific incorporation site, and at least one of the two paired guide RNAs is an atgRNA that further includes a reverse transcriptase (RT) template that comprises at least a portion of the first integration recognition site, and the atgRNAs collectively encode the entirety of the first integration recognition site, whereby the first integration recognition site is site-specifically incorporated into the genome of the pleiopluripotent cell.
3. The method of claim 2, wherein the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is a first atgRNA that further includes a first RT template that comprises at least a portion of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a second atgRNA that further includes a second RT template that comprises at least a portion of the first integration recognition site, wherein the first atgRNA and the second atgRNAs collectively encode the entirety of the first integration recognition site.
4. The method of claim 2, wherein the at least first pair of guide RNAs comprise: (i) the first of the two paired guide RNAs is an atgRNA that further includes an RT template that comprises at least a portion of the first integration recognition site, wherein the atgRNA encodes the entirety of the first integration recognition site; and (ii) a second of the two paired guide RNAs is a nicking gRNA.
5. The method of any one of claims 1-4, further comprising incorporating a plurality of integration recognition sites.
6. The method of any one of claims 1-5, further comprising: (b) integrating at least a first donor polynucleotide template into the pleiopluripotent cell genome at the first incorporated integration recognition site, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a second generation pleiopluripotent cell.
7. The method of any one of claims 1-6, wherein steps (a) and (b) are performed concurrently.
8. The method of any one of claims 1-6, wherein step (a) is performed prior to step (b).
9. A method of generating a pleiopluripotent cell, the method comprising: (a) integrating, into the genome of the pleiopluripotent cell of any one of claims 1-8 at the first incorporated recognition site, at least a first donor polynucleotide template, by introducing into the cell: (i) the first donor polynucleotide template, wherein the first donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the at least one incorporated genomic integration recognition sites by the integrase; thereby producing a second generation pleiopluripotent cell.
10. The method of any one of claims 1-9, further comprising (b) site-specifically incorporating a second integration recognition site into the genome of the pleiopluripotent cell, thereby generating a third generation pleiopluripotent cell.
11. The method of any one of claims 1-10, further comprising: (c) integrating a second donor polynucleotide template into the pleiopluripotent cell genome at the second incorporated integration recognition site, by delivering into the cell: (i) the second donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more integration recognition sites orthogonal to the second integration recognition site, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the second incorporated genomic integration recognition sites by the integrase; thereby producing a fourth generation pleiopluripotent cell line.
12. The method of any one of claims 9-11, wherein steps (a), (b), and (c) are performed concurrently.
13. The method of any one of claims 9-11, wherein step (a) is performed prior to steps (b) and (c), wherein steps (b) and (c) are performed concurrently or step (b) is performed prior to step (c).
14. The method of any one of claims 1-11, wherein the step of site-specifically incorporating the first integration recognition site and the step of site-specifically incorporating the second integration recognition site are performed concurrently.
15. The method of any one of claims 3-14, wherein the first RT template encodes a first single-stranded DNA sequence and the second RT template encodes a second single- stranded DNA sequence.
16. The method of any one of claims 3-15, wherein the first single-stranded DNA sequence comprises a complementary region with the first single-stranded DNA sequence.
17. The method of any one of claims 3-16, wherein the first single-stranded DNA sequence and the first single-stranded DNA sequence form a duplex.
18. The method of any one of claims 16 or 17, wherein the complementary region is 5 or more consecutive bases.
19. The method of any one of claims 16 or 17, wherein the complementary region is 10 or more consecutive cases.
20. The method of any one of claims 16 or 17, wherein the complementary region is 20 or more consecutive bases.
21. The method of any one of claims 16 or 17, wherein the complementary region is 30 or more consecutive bases.
22. The method of any one of claims 2-21, wherein at least one of the two paired guide RNAs has a chemical modification.
23. The method of any one of claims 2-22, wherein the paired guide RNAs each have a chemical modification.
24. The method of any one of claims 1-23, wherein at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a safe harbor locus.
25. The method of any one of claims 1-23, wherein at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell.
26. The method of claim 5, wherein at least one of the at least first integration recognition sites is incorporated into the genome at a plurality of loci, wherein disruption of at least one of the loci is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell.
27. The method of any one of claims 1-26, wherein at least one of the at least first integration recognition sites is incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
28. The method of any one of claims 1-27, wherein the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
29. The method of any one of claims 25-28, wherein the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
30. The method of any one of claims 25-29, wherein the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus.
31. The method of claim 30, wherein the locus is the B2M locus.
32. The method of claim 30, wherein the locus is the CIITA locus.
33. The method of any one of claims 1-32, wherein the introducing step is performed by electroporation.
34. The method of claim 33, wherein introducing comprises electroporating a gene editor protein or a polynucleotide encoding a gene editor protein, a prime editor fusion protein or a polynucleotide encoding a prime editor fusion protein, at least a first pair or guide RNAs, the donor polynucleotide template, or a combination thereof into the pleiopluripotent cell occurs using one or more of a recombinant adenovirus, helper dependent adenovirus, AAV, lentivirus, HSV, anellovirus, retrovirus, Doggybone DNA, minicircle, plasmid, miniDNA, nanoplasmid, exosome, fusosome, mRNA, RNP, or lipid nanoparticle.
35. The method of any one of claims 1-32, wherein the introducing step is performed by transfection.
36. The method of claim 35, wherein introducing comprises transfecting mRNA encoding the gene editor protein, the prime editor fusion protein, the at least first pair of guide gRNAs, the donor polynucleotide template, or a combination thereof into the pleiopluripotent cell, wherein the donor polynucleotide template is selected from a mini circle, a nanoplasmid, and a miniDNA.
37. The method of any one of claims 1-36, wherein at least one of the at least first integration recognition sites is specific for a serine integrase.
38. The method of any one of claims 1-37, wherein at least one of the at least first integration recognition sites is an attB or attP site.
39. The method of any one of claims 1-38, wherein at least one of the at least first integration recognition sites is a modified attB or attP site.
40. The method of any one of claims 1-39, wherein at least one of the at least first integration recognition sites is specific for BxB1 or a modified BxB1.
41. The method of any one of claims 1-40, wherein at least one of the at least first integration recognition sites is comprised of 38 or 46 nucleotides.
42. The method of any one of claims 6-41, wherein the first donor polynucleotide template encodes one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof.
43. The method of claim 42, wherein the inducing the at least one of the one or more inducible suicide genes targets the pleiopluripotent cell or a cell matured from the pleiopluripotent cell for cell death.
44. The method of claim 42 or 43, wherein expression of at least of the one or more inducible suicide gene is driven by a pluripotent stem cell-specific promoter.
45. The method of claim 42 or 43, wherein expression of at least one of the one or more inducible suicide gene is driven by a constitutive promoter.
46. The method of claim 42 or 43, wherein expression of at least one of the one or more inducible suicide gene is driven by an inducible promoter.
47. The method of any one of claims 42-46, wherein the one or more inducible suicide genes is selected from: caspase9, cytosine deaminase, and thymidine kinase.
48. The method of claim 47, wherein the one or more inducible suicide genes is a controllable caspase9.
49. The method of claim 48, wherein AP20187 (or an analog thereof) controls activity of Caspase9 or AP21967 (or an analog thereof) controls activity of Caspase9.
50. The method of any one of claims 42-49, further comprising a second inducible suicide gene, wherein the second inducible suicide gene comprises a thymidine kinase.
51. The method of claim 50, wherein the polynucleotide encoding for the thymidine kinase is operably linked to the controllable capase9 protein using a 2A self-cleaving peptide or an internal ribosome entry site.
52. The method of any one of claims 42-51, wherein the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells.
53. The method of claim 52, wherein the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells by inhibiting, blocking, or preventing recognition of the pleiopluripotent cell by macrophages, T-cells, Natural Killer (NK)-cells, antigen-presenting cells, or a combination thereof.
54. The method of claim 52 or 53, wherein the one or more exogenous polypeptides capable of modulating an immune response are capable of serving as a safety switch target.
55. The method of any one of claims 42-54, wherein expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a pluripotent stem cell-specific promoter.
56. The method of any one of claims 42-54, wherein expression of least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a constitutive promoter.
57. The method of any one of claims 42-54, wherein expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by an inducible promoter.
58. The method of any one of claims 42-57, wherein the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL-1, B2M-HLA- E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR.
59. The method of claim 58, wherein the donor polynucleotide template encodes a CD47 polypeptide, a PDL1 polypeptide, and a B2M-HLA-E polypeptide.
60. The method of claim 59, wherein each of the sequences coding for CD47, PDL1 and B2M-HLA-E are separated by a sequence coding for a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof.
61. The method of any one of claims 42-60, wherein the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag.
62. The method of any one of claims 6-61, wherein the donor polynucleotide template further comprises an additional orthogonal integrase target recognition site.
63. The method of any one of claims 10-62, wherein the second integration recognition site is site-specifically incorporated into a safe harbor locus.
64. The method of any one of claims 10-63, wherein the second integration recognition site is different from the at least first integration recognition attB or attP site.
65. The method of any one of claims 10-64, wherein the second integration recognition site is specific for BxB1 or a modified BxB1.
66. The method of any one of claims 10-65, wherein the second integration recognition sites is comprised of 38 or 46 nucleotides.
67. The method of any one of claims 11-66, wherein a second donor polynucleotide template is integrated into the pleiopluripotent cell genome at the second integration recognition site site-specifically incorporated into the pleiopluripotent cell genome.
68. The method of any one of claims 11-67, wherein the second donor polynucleotide template encodes one or more therapeutic agents.
69. The method of claim 68, wherein the one or more therapeutic agents is selected from: transcription factors, receptors, signaling molecules, secreted proteins, cytokines, enzymes, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, and persistence and/or survival of the iPSCs or cells derived from the iPSC.
70. The method of any one of claims 11-69, the one or more therapeutic agents is a HLA class I protein.
71. The method of any one of claims 11-70, the one or more therapeutic agents is a HLA class II protein.
72. The method of any one of claims 11-71, wherein the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents.
73. The method of any one of claims 11-71, wherein the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agents.
74. The method of any one of claims 11-71, wherein the second donor polynucleotide template comprises an inducible promoter operably linked to at least one of the one or more therapeutic agents.
75. The method of any one of claims 6-74, wherein the first donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 1 kilobase (kb), at least 2 kb, at least 3kb, at least4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, at least 30kb, at least 31kb, at least 32kb, at least 33kb, at least 34kb, or at least 35kb or more.
76. The method of claim 75, wherein the first donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 30kb.
77. The method of any one of claims 11-76, wherein the second donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 1 kilobase (kb), at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 11kb, at least 12kb, at least 13kb, at least 14kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, at least 30kb, at least 31kb, at least 32kb, at least 33kb, at least 34kb, or at least 35kb or more.
78. The method of claim 77, wherein the second donor polynucleotide template integrated into the pleiopluripotent cell genome is at least 30kb.
79. The method of any one of claims 1-78, wherein the pleiopluripotent cell is a pluripotent stem cell.
80. The method of any one of claims 1-79, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
81. The method of any one of claims 1-80, wherein the pluripotent stem cell is an induced pluripotent stem cell.
82. The method of any one of claims 1-81, wherein the induced pluripotent stem cell is a human induced pluripotent stem cell.
83. The method of any one of claims 6-82, further comprising site-specifically excising from the genome of the pleiopluripotent cell the first donor polynucleotide template, the second donor polynucleotide template, or both.
84. The method of claim 83, wherein the site-specifically excising is effected by introducing into the pleiopluripotent cell an integrase that recognizes the one or more orthogonal integration recognition sites in the first donor polynucleotide template, the second donor polynucleotide template, or both.
85. The method of any one of claims 1-84, further comprising cryopreserving the pleiopluripotent cell or a population thereof.
86. A pleiopluripotent cell or population thereof generated using the method any one of claims 1-85.
87. A second generation pleiopluripotent cell generated using the method of any of the one of the claims 6-85.
88. A third generation pleiopluripotent cell generated using the method of any of the one of the claims 10-85.
89. A fourth generation pleiopluripotent cell generated using the method of any of the one of the claims 11-85.
90. A pleiopluripotent cell, comprising: at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome.
91. A pleiopluripotent cell comprising: a donor polynucleotide template comprised of one or more orthogonal integration recognition site integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
92. The pleiopluripotent cell of claim 90 or 91, wherein at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a safe harbor locus.
93. The pleiopluripotent cell of claim 90 or 91, wherein at least one of the at least first integration recognition sites is site-specifically incorporated within a locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell.
94. The pleiopluripotent cell of claim 90 or 91, wherein at least one of the at least first integration recognition sites is site-specifically incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
95. The pleiopluripotent cell of claim 94, wherein the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
96. The pleiopluripotent cell of any one of claims 93-95, wherein the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
97. The pleiopluripotent cell of any one of claims 90-96, wherein the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA- DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus.
98. The pleiopluripotent cell of claim 97, wherein the locus is the B2M locus.
99. The pleiopluripotent cell of claim 97, wherein the locus is the CIITA locus.
100. The pleiopluripotent cell of any one of claims 90-99, wherein at least one of the at least first integration recognition sites is specific for a serine integrase.
101. The pleiopluripotent cell of any one of claims 90-100, wherein at least one of the at least first integration recognition sites is an attB or attP site.
102. The pleiopluripotent cell of any one of claims 90-101, wherein at least one of the at least first integration recognition sites is a modified attB or attP site.
103. The pleiopluripotent cell of any one of claims 90-102, wherein at least one of the at least first integration recognition sites is specific for BxB1 or a modified BxB1.
104. The pleiopluripotent cell of any one of claims 90-103, wherein at least one of the at least first integration recognition site is comprised of 38 or 46 nucleotides.
105. The pleiopluripotent cell of any one of claims 90-104, wherein the donor polynucleotide template encodes one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof.
106. The pleiopluripotent cell of claim 105, wherein inducing at least one of the one or more inducible suicide genes targets the pleiopluripotent cell or a cell matured from the pleiopluripotent cell for cell death.
107. The pleiopluripotent cell of claim 105 or 106, wherein expression of at least one of the one or more inducible suicide gene is driven by a pluripotent stem cell-specific promoter.
108. The pleiopluripotent cell of claim 105 or 106, wherein expression of at least one of the one or more inducible suicide gene is driven by a constitutive promoter.
109. The pleiopluripotent cell of claim 105 or 106, wherein expression of at least one of the one or more inducible suicide gene is driven by an inducible promoter.
110. The pleiopluripotent cell of any one of claims 105-109, wherein the one or more inducible suicide genes is selected from: caspase9, cytosine deaminase, and thymidine kinase.
111. The pleiopluripotent cell of claim 110, wherein the one or more inducible suicide genes is a controllable caspase9.
112. The pleiopluripotent cell of claim 111, wherein AP20187 (or analog thereof) controls activity of Caspase9 or AP21967 (or analog thereof) controls activity of Caspase9.
113. The pleiopluripotent cell of any one of claims 105-112, further comprising a second inducible suicide gene.
114. The pleiopluripotent cell of claim 113, wherein the second inducible suicide gene comprises a thymidine kinase.
115. The pleiopluripotent cell of claim 114, wherein the polynucleotide encoding for the thymidine kinase is operably linked to the controllable capase9 protein using a 2A self- cleaving peptide or an internal ribosome entry site.
116. The pleiopluripotent cell of any one of claims 105-115, wherein the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells.
117. The pleiopluripotent cell of claim 116, wherein the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells by inhibiting, blocking, or preventing recognition of the PC-derived cell by macrophages, T-cells, Natural Killer (NK)-cells, antigen-presenting cells, or a combination thereof.
118. The pleiopluripotent cell of any one of claims 105-117, wherein the one or more exogenous polypeptides capable of modulating an immune response are capable of serving as a safety switch target.
119. The pleiopluripotent cell of any one of claims 105-118, wherein expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a pluripotent stem cell-specific promoter.
120. The pleiopluripotent cell of any one of claims 105-118, wherein expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by a constitutive promoter.
121. The pleiopluripotent cell of any one of claims 105-118, wherein expression of at least one of the one or more exogenous polypeptides capable of modulating an immune response is driven by an inducible promoter.
122. The pleiopluripotent cell of any one of claims 105-121, wherein the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL-1, B2M-HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof.
123. The pleiopluripotent cell of claim 122, wherein the donor polynucleotide template encodes a CD47, PDL1, and B2M-HLA-E.
124. The pleiopluripotent cell of claim 123, wherein the polynucleotide template encoding CD47, PDL1 and B2M-HLA-E are separated by a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof.
125. The pleiopluripotent cell of any one of claims 105-124, wherein the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag.
126. The pleiopluripotent cell of claim 125, wherein the tag is an ALFA tag.
127. The pleiopluripotent cell of any one of claims 90-125, comprising two integration recognition sites, three integration recognition sites, four integration recognition sites, five integration recognition sites, six integration recognition sites, seven integration recognition sites, eight integration recognition sites, nine integration recognition sites, or ten or more integration recognition sites.
128. The pleiopluripotent cell of any one of claims 90-127, further comprising a second integration recognition site site-specifically incorporated into the pleiopluripotent cell genome.
129. The pleiopluripotent cell of claim 128, wherein the second integration recognition sites is site-specifically incorporated into a safe harbor locus.
130. The pleiopluripotent cell of claim 128, wherein the second integration recognition site is site-specifically incorporated within a second locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell.
131. The pleiopluripotent cell of claim 130, wherein the second integration recognition site is site-specifically incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
132. The pleiopluripotent cell of claim 130 or 131, wherein the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
133. The pleiopluripotent cell of any one of claims 130-132, wherein the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
134. The pleiopluripotent cell of any one of claim 130-133, wherein the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA- DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, and RFXANK locus.
135. The pleiopluripotent cell of claim 134, wherein the locus is the B2M locus.
136. The pleiopluripotent cell of claim 134, wherein the locus is the CIITA locus.
137. The pleiopluripotent cell of any one of claims 128-136, wherein at least one of the at least second integration recognition sites is specific for a serine integrase.
138. The pleiopluripotent cell of any one of claims 128-137, wherein the second integration recognition sites is different from the at least first integration recognition attB or attP site.
139. The pleiopluripotent cell of any one of claims 128-137, wherein the second integration recognition sites is a modified attB or attP site.
140. The pleiopluripotent cell of any one of claims 128-139, wherein the second integration recognition site is specific for BxB1 or a modified BxB1.
141. The pleiopluripotent cell of any one of claims 128-140, wherein the second integration recognition sites is comprised of 38 or 46 nucleotides.
142. The pleiopluripotent cell of any one of claims 128-141, wherein the second donor polynucleotide template encodes one or more therapeutic agents.
143. The pleiopluripotent cell of any one of claim 142, wherein the one or more therapeutic agents is selected from: transcription factors, receptors, signaling molecules, secreted proteins, cytokines, enzymes, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, and persistence and/or survival of the iPSCs or cells derived from the iPSC.
144. The pleiopluripotent cell of claim 142 or 143, wherein the one or more therapeutic agents is a HLA class I proteins.
145. The pleiopluripotent cell of any one of claims 142 or 143, wherein the one or more therapeutic agents is a HLA class II proteins.
146. The pleiopluripotent cell of any one of claims 128-145, wherein the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents.
147. The pleiopluripotent cell of any one of claims 128-146, wherein the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agent.
148. The pleiopluripotent cell of any one of claims 128-147, wherein the second donor polynucleotide template comprises an inducible promoter operably linked to at least one of the one or more therapeutic agent.
149. The pleiopluripotent cell of any one of claims 90-148, wherein the pleiopluripotent cell is a pluripotent stem cell.
150. The pleiopluripotent cell of any one of claims 90-149, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
151. The pleiopluripotent cell of any one of claims 90-150, wherein the pluripotent stem cell is an induced pluripotent stem cell.
152. The pleiopluripotent cell of any one of claims 90-151, wherein the induced pluripotent stem cell is a human induced pluripotent stem cell.
153. A composition comprising a clonal population of pleiopluripotent cells of claims 86-152.
154. A pharmaceutical composition comprising a clonal population of pleiopluripotent cells of claims 86-152 and a pharmaceutically acceptable excipient or carrier.
155. A pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site is incorporated site-specifically into the PC-derived cell genome.
156. A pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
157. A pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a hematopoietic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site is incorporated site-specifically into the PC-derived cell genome.
158. A pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a hematopoietic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
159. The PC-derived cell or a population thereof of claim 157 or 158, the hematopoietic cell is selected from: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells.
160. A pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a neuronal cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site integration recognition sites incorporated site-specifically into the PSC-derived cell genome.
161. A pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a neuronal cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
162. The PC-derived cell or a population thereof of claim 160 or 161, wherein the neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron.
163. A pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a cardiac cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site incorporated site-specifically into the PSC-derived cell genome.
164. A pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a cardiac cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
165. The PC-derived cell or a population thereof of any one of claims 163 or 164, wherein the cardiac cell is selected from: a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte.
166. A pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a pancreatic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises at least a first integration recognition site incorporated site-specifically into the PC-derived cell genome.
167. A pleiopluripotent cell (PC)-derived cell or a population thereof, wherein (i) the PC-derived cell is a pancreatic cell differentiated from a pleiopluripotent cell; and (ii) the PC-derived cell comprises a first donor polynucleotide template, comprised of one or more orthogonal integration recognition site, integrated into the pleiopluripotent cell genome at an at least first integration recognition site, wherein the at least first integration recognition site is site-specifically incorporated into the pleiopluripotent cell genome.
168. The PC-derived cell or a population thereof of claim 166 or 167, wherein the pancreatic cell is selected from a pancreatic progenitor cell, pancreatic endoderm, a endocrine pancreatic cell, an exocrine pancreatic cell, a islet progenitor, and a beta cell.
169. The PC-derived cell or population thereof of any one of claims 155-168, wherein the PC- derived cell further comprises two integration recognition sites, three integration recognition sites, four integration recognition sites, five integration recognition sites, six integration recognition sites, seven integration recognition sites, eight integration recognition sites, nine integration recognition sites, or ten or more integration recognition sites.
170. The PC-derived cell or population thereof of any one of claims 155-169, wherein at least one of the at least first integration recognition sites is incorporated into the pleiopluripotent cell genome at a safe harbor locus.
171. The PC-derived cell or population thereof of any one of claims 155-169, wherein at least one of the at least first integration recognition sites is site-specifically incorporated within a locus, disruption of which is capable of reducing allogeneic immune response to the PC-derived cell.
172. The PC-derived cell or a population thereof of any one of claims 155-170, wherein at least one of the at least first integration recognition sites is incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
173. The PC-derived cell or a population thereof of claim 171 or 172, wherein the pleiopluripotent cell comprises deletion or reduced expression of one or more HLA Class I proteins, one or more HLA Class II proteins, or a combination thereof.
174. The PC-derived cell or a population thereof of any one of claims 171-173, wherein the locus is in a chromosomal region selected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.
175. The PC-derived cell or a population thereof of any one of claims 155-174, wherein the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA-DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus.
176. The PC-derived cell or population thereof of claim 175, wherein the locus is the B2M locus.
177. The PC-derived cell or population thereof of claim 175, wherein the locus is the CIITA locus.
178. The PC-derived cell or population thereof of any one of claims 155-177, wherein at least one of the at least first recognition sites is specific for a serine integrase.
179. The PC-derived cell or population thereof of any one of claims 155-178, wherein at least one of the at least first integration recognition sites is an attB or attP site.
180. The PC-derived cell or population thereof of any one of claims 155-179, wherein at least one of the at least first integration recognition sites is a modified attB or attP site.
181. The PC-derived cell or population thereof of any one of claims 155-180, wherein at least one of the at least first integration recognition site is specific for BxB1 or a modified BxB1.
182. The PSC-derived cell or population thereof of any one of claims 155-181, wherein at least one of the at least first integration recognition sites is comprised of 38 or 46 nucleotides.
183. The PC-derived cell or population thereof of any one of claims 155-182, wherein the first donor polynucleotide template encodes one or more inducible suicide genes, one or more exogenous polypeptides capable of modulating an immune response, or a combination thereof.
184. The PC-derived cell of claim 183, inducing at least one of the one or more inducible suicide genes targets the pleiopluripotent cell or a cell matured from the pleiopluripotent cell for cell death.
185. The PC-derived cell or population thereof of claim 183 or 184, wherein expression of at least one of the one or more inducible suicide gene is driven by a pluripotent stem cell- specific promoter.
186. The PC-derived cell or population thereof of claim 183 or 184, wherein expression of the at least one of the one or more inducible suicide genes is driven by a constitutive promoter.
187. The PC-derived cell or population thereof of claim 183 or 184, wherein expression of the at least one of the one or more inducible suicide genes is driven by an inducible promoter.
188. The PC-derived cell or population thereof of any one of claims 183-187, wherein the one or more inducible suicide genes is select from: caspase9, cytosine deaminase, and thymidine kinase.
189. The PC-derived cell or population thereof of claim 188, wherein the one or more inducible suicide genes is a controllable caspase9.
190. The PC-derived cell or population thereof of claim 189, wherein AP20187 (or analog thereof) controls activity of Caspase9 or AP21967 (or analog thereof) controls activity of Caspase9.
191. The PC-derived cell or population thereof of claim 189, further comprising a second inducible suicide gene,
192. The PC-derived cell or population thereof of claim 191, wherein the polynucleotide encoding for the second inducible suicide gene is operably linked to the controllable capase9 protein using a 2A self-cleaving peptide or an internal ribosome entry site.
193. The PC-derived cell or population thereof of claim 191 or 192, wherein the second inducible suicide gene comprises a thymidine kinase.
194. The PC-derived cell or population thereof of any one of claims 183-193, wherein the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells.
195. The PC-derived cell or population thereof of claim 194, wherein the one or more exogenous polypeptides capable of modulating an immune response prevents an immune response to the pleiopluripotent cells by inhibiting, blocking, or preventing recognition of the PC-derived cell by macrophages, T-cells, Natural Killer (NK)-cells, antigen- presenting cells, or a combination thereof.
196. The PC-derived cell or population thereof of claims 194 or 195, wherein the one or more exogenous polypeptides capable of modulating an immune response are capable of serving as a safety switch target.
197. The PC-derived cell or population thereof of any one of claims 183-196, wherein expression of at least one of the one or more exogenous polypeptide capable of modulating an immune response is driven by a pluripotent stem cell-specific promoter.
198. The PC-derived cell or population thereof of any one of claims 183-196, wherein expression of at least one of the one or more exogenous polypeptide capable of modulating an immune response is driven by a constitutive promoter.
199. The PC-derived cell or population thereof of any one of claims 183-196, wherein expression of at least one of the one or more exogenous polypeptide capable of modulating an immune response is driven by an inducible promoter.
200. The PC-derived cell or population thereof of any one of claims 183-199, wherein the one or more exogenous polypeptides capable of modulating an immune response is selected from: CD47, PDL-1, B2M-HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD137, CD80, and A2AR, or a combination thereof.
201. The PC-derived cell or population thereof of claim 200, wherein the donor polynucleotide template encodes a CD47, PDL1, and B2M-HLA-E.
202. The PC-derived cell or population thereof of claim 201, wherein the polynucleotide template encoding CD47, PDL1 and B2M-HLA-E are separated by a 2A self-cleaving peptide, an internal ribosome entry site, or a combination thereof.
203. The PC-derived cell or population thereof of any one of claims 183-202, wherein the donor polynucleotide template further comprises a polynucleotide sequence encoding a tag.
204. The PC-derived cell or population thereof of claim 203, wherein the tag is an ALFA tag.
205. The PC-derived cell or population thereof of any one of claims 155-204, further comprising a second integration recognition site site-specifically incorporated into the pleiopluripotent cell genome.
206. The PC-derived cell or population thereof of claim 205, wherein the second integration recognition sites is site-specifically incorporated into a safe harbor locus.
207. The PC-derived cell or population thereof of claim 205, wherein the second integration recognition site is site-specifically incorporated within a second locus, disruption of which is capable of reducing allogeneic immune response to the pleiopluripotent cell or a cell matured from the pleiopluripotent cell.
208. The PC-derived cell or population thereof of claim 207, wherein the second integration recognition site is site-specifically incorporated into the genome at a locus required for surface expression of HLA class I proteins, HLA class II proteins, or a combination thereof.
209. The PC-derived cell or a population thereof of claim 207 or 208, wherein the locus is selected from B2M locus, CIITA locus, HLA-A locus, HLA-B locus, HLA-C locus, HLA- DR locus, HLA-DP locus, HLA-DQ locus, TAP1 locus, TAP2 locus, LRC5 locus, CCR5 locus, CXCR4 locus, PD1 locus, LAG3 locus, TIM3 locus, RFXAP locus, RFX5 locus, or RFXANK locus.
210. The PC-derived cell or population thereof of claim 209, wherein the locus is the B2M locus.
211. The PC-derived cell or population thereof of claim 209, wherein the locus is the CIITA locus.
212. The PC-derived cell or population thereof of any one of claims 205-211, wherein the second integration recognition site is specific for a serine integrase.
213. The PC-derived cell or population thereof of any one of claims 205-212, wherein the second integration recognition sites is different from the at least first integration recognition attB or attP site.
214. The PC-derived cell or population thereof of any one of claims 205-213, wherein the second integration recognition site is an attB or attP site.
215. The PC-derived cell or population thereof of any one of claims 205-214, wherein the second integration ecognition sites is a modified attB or attP site.
216. The PC-derived cell or population thereof of any one of claims 205-215, wherein the second integration ecognition site is specific for BxB1 or a modified BxB1.
217. The PC-derived cell or population thereof of any one of claims 205-216, wherein the second integration recognition sites is comprised of 38 or 46 nucleotides.
218. The PC-derived cell or population thereof of any one of claims 205-217, wherein the donor polynucleotide template encodes one or more orthogonal integration recognition sites.
219. The PC-derived cell or population thereof of any one of claims 205-218, wherein a second donor polynucleotide template is integrated into the PC-derived cell genome at the second integration recognition site site-specifically incorporated into the PC-derived cell genome.
220. The PC-derived cell or population thereof of any one of claims 205-219, wherein the first donor polynucleotide template is encodes one or more therapeutic agents.
221. The PC-derived cell or population thereof of claim 220, wherein the one or more therapeutic agents is selected from: transcription factors, receptors, signaling molecules, secreted proteins, cytokines, enzymes, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, and persistence and/or survival of the iPSCs or cells derived from the iPSC.
222. The PC-derived cell or population thereof of claim 220 or 221, wherein the one or more therapeutic agents is a HLA class I proteins.
223. The PC-derived cell or population thereof of claim 220 or 221, wherein the one or more therapeutic agents is a HLA class II proteins.
224. The PC-derived cell or population thereof of any one of claims 220-223, wherein the second donor polynucleotide template comprises a pluripotent stem cell-specific promoter operably linked to at least one of the one or more therapeutic agents.
225. The PC-derived cell or population thereof of any one of claims 220-223, wherein the second donor polynucleotide template comprises a constitutive promoter operably linked to at least one of the one or more therapeutic agent.
226. The PC-derived cell or population thereof of any one of claims 220-223, wherein the second donor polynucleotide template comprises an inducible promoter operably linked to at least one therapeutic agent.
227. The PC-derived cell or population thereof of any one of claims 155-226, wherein the PC- derived cell or population thereof are human cells.
228. A composition comprising a population of PC-derived cells of claims 155-227.
229. A pharmaceutical composition comprising a population of PC-derived cells of claims 155-227 and a pharmaceutically acceptable excipient or carrier.
230. A method of treating or ameliorating or preventing a disease or condition in a subject, comprising administering a therapeutically effective amount of the PC-derived cell or population thereof of any one of claims 155-227 composition of claim 228 or the pharmaceutical composition of claim 229.
231. The method of treating or ameliorating or preventing a disease according to claim 230, wherein the disease is a cancer.
232. The method of treating or ameliorating or preventing a disease according to claim 230, wherein the disease is a muscular and/or the condition is muscle degeneration or muscle injury.
233. The method of treating or ameliorating or preventing a disease according to claim 230, wherein the disease is a neuronal disease and/or the condition is neuron degeneration
234. The method of treating or ameliorating or preventing a disease according to claim 230, wherein the disease is associated with the pancreas.
235. A method of using a pleiopluripotent cell having at least a first integration recognition site site-specifically incorporated into the pleiopluripotent cell genome, the method comprising: integrating a first donor polynucleotide template into the pleiopluripotent cell genome by introducing into the pleiopluripotent cell: i) the donor polynucleotide template, wherein the donor polynucleotide template is comprised of one or more orthogonal integration recognition sites, and (ii) an integrase, whereby the donor polynucleotide is integrated into the pleiopluripotent cell at the at least one incorporated genomic integration recognition sites by the integrase.
236. The method of claim 235, further comprising selecting the pleiopluripotent cells having the first donor polynucleotide template site-specifically integrated into the genome.
237. The method of claim 235 or 236, further comprising expanding the pleiopluripotent cells in a de-differentiated state.
238. The method of any one of claims 235-237, further comprising cryopreserving the pleiopluripotent cells.
239. The method of any one of claim 235-238, further comprising, further comprising directing differentiation of the modified pleiopluripotent cell to a hematopoietic cell.
240. The method of claim 239, wherein the hematopoietic cell is selected from: hematopoietic stem and progenitor cells (HSC), pre-HSC, hematopoietic multipotent progenitor (MPP) cells, pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells.
241. The method of any one of claims 235-240, further comprising directing differentiation of the modified pleiopluripotent cell to a neuronal cell.
242. The method of claims 241, wherein the neuronal cell is selected from: a neural progenitor cell (NPC), a forebrain neuron, a dopaminergic neuron, a motor neuron, and a sensory neuron.
243. The method of any one of claims 235-240, further comprising directing differentiation of the modified pleiopluripotent cell to a cardiac cell.
244. The method of claim 243, wherein the cardiac cell is selected from: a cardiac mesodermal cell, a cardiac progenitor cell, and a cardiomyocyte.
245. The method of any one of claims 235-240, further comprising directing differentiation of the modified pleiopluripotent cell to a pancreatic cell.
246. The method of claim 245, wherein the cardiac cell is selected from: pancreatic progenitor cell, pancreatic endoderm, an endocrine pancreatic cell, an exocrine pancreatic cell, a islet progenitor, and a beta cell.
247. The method of any one of claim 239-246, further comprising administering the PC- derived cells or a population thereof to a patient in need thereof.
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