CN115768886A - 一种基因组编辑系统及方法 - Google Patents
一种基因组编辑系统及方法 Download PDFInfo
- Publication number
- CN115768886A CN115768886A CN202080101503.1A CN202080101503A CN115768886A CN 115768886 A CN115768886 A CN 115768886A CN 202080101503 A CN202080101503 A CN 202080101503A CN 115768886 A CN115768886 A CN 115768886A
- Authority
- CN
- China
- Prior art keywords
- sequence
- cas12f
- gene
- nuclease
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000010362 genome editing Methods 0.000 title claims abstract description 82
- 238000000034 method Methods 0.000 title claims abstract description 38
- 101710163270 Nuclease Proteins 0.000 claims abstract description 82
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 73
- 108020005004 Guide RNA Proteins 0.000 claims abstract description 46
- 230000008685 targeting Effects 0.000 claims abstract description 30
- 230000014509 gene expression Effects 0.000 claims abstract description 24
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 12
- 108020004414 DNA Proteins 0.000 claims description 81
- 238000003776 cleavage reaction Methods 0.000 claims description 41
- 230000007017 scission Effects 0.000 claims description 41
- 239000012634 fragment Substances 0.000 claims description 32
- 102000053602 DNA Human genes 0.000 claims description 20
- 241000182988 Assa Species 0.000 claims description 17
- 238000003780 insertion Methods 0.000 claims description 14
- 230000037431 insertion Effects 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 230000035897 transcription Effects 0.000 claims description 12
- 238000013518 transcription Methods 0.000 claims description 12
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 9
- 238000012224 gene deletion Methods 0.000 claims description 8
- 230000035772 mutation Effects 0.000 claims description 8
- 150000007523 nucleic acids Chemical class 0.000 claims description 8
- 102000039446 nucleic acids Human genes 0.000 claims description 7
- 108020004707 nucleic acids Proteins 0.000 claims description 7
- 241000186581 Clostridium novyi Species 0.000 claims description 6
- 108091026890 Coding region Proteins 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 241000186394 Eubacterium Species 0.000 claims description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 4
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 claims 2
- 241001598819 Acidibacter Species 0.000 claims 1
- 241001408449 Asca Species 0.000 claims 1
- 241001531189 [Eubacterium] siraeum Species 0.000 claims 1
- 238000005520 cutting process Methods 0.000 abstract description 13
- 238000000338 in vitro Methods 0.000 abstract description 12
- 238000002474 experimental method Methods 0.000 abstract description 11
- 239000013612 plasmid Substances 0.000 description 99
- 210000004027 cell Anatomy 0.000 description 58
- 238000003752 polymerase chain reaction Methods 0.000 description 39
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 36
- 238000006243 chemical reaction Methods 0.000 description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 27
- 102000040430 polynucleotide Human genes 0.000 description 25
- 108091033319 polynucleotide Proteins 0.000 description 25
- 239000002157 polynucleotide Substances 0.000 description 25
- 239000000047 product Substances 0.000 description 25
- 241000588724 Escherichia coli Species 0.000 description 24
- 230000001580 bacterial effect Effects 0.000 description 24
- 241000588747 Klebsiella pneumoniae Species 0.000 description 21
- 238000010276 construction Methods 0.000 description 20
- 239000000758 substrate Substances 0.000 description 20
- 230000000694 effects Effects 0.000 description 19
- 108091033409 CRISPR Proteins 0.000 description 18
- 238000002360 preparation method Methods 0.000 description 18
- 125000006850 spacer group Chemical group 0.000 description 18
- 239000013613 expression plasmid Substances 0.000 description 16
- 101150066555 lacZ gene Proteins 0.000 description 15
- 241000894006 Bacteria Species 0.000 description 13
- 239000007788 liquid Substances 0.000 description 13
- 238000012163 sequencing technique Methods 0.000 description 13
- 239000011780 sodium chloride Substances 0.000 description 13
- XZNUGFQTQHRASN-XQENGBIVSA-N apramycin Chemical compound O([C@H]1O[C@@H]2[C@H](O)[C@@H]([C@H](O[C@H]2C[C@H]1N)O[C@@H]1[C@@H]([C@@H](O)[C@H](N)[C@@H](CO)O1)O)NC)[C@@H]1[C@@H](N)C[C@@H](N)[C@H](O)[C@H]1O XZNUGFQTQHRASN-XQENGBIVSA-N 0.000 description 12
- 229950006334 apramycin Drugs 0.000 description 12
- 108090000765 processed proteins & peptides Proteins 0.000 description 12
- 102000004196 processed proteins & peptides Human genes 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 238000010354 CRISPR gene editing Methods 0.000 description 11
- 210000005260 human cell Anatomy 0.000 description 11
- 229920001184 polypeptide Polymers 0.000 description 11
- 239000007787 solid Substances 0.000 description 11
- 230000003321 amplification Effects 0.000 description 10
- 230000001404 mediated effect Effects 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 238000003199 nucleic acid amplification method Methods 0.000 description 10
- 239000007795 chemical reaction product Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 108020004705 Codon Proteins 0.000 description 8
- 230000007018 DNA scission Effects 0.000 description 8
- 238000012986 modification Methods 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 230000010474 transient expression Effects 0.000 description 8
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 7
- 238000012217 deletion Methods 0.000 description 7
- 230000037430 deletion Effects 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 229930027917 kanamycin Natural products 0.000 description 7
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 7
- 229960000318 kanamycin Drugs 0.000 description 7
- 229930182823 kanamycin A Natural products 0.000 description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- 108700004991 Cas12a Proteins 0.000 description 6
- 241001052560 Thallis Species 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- 230000000295 complement effect Effects 0.000 description 6
- 101150101290 dhaK gene Proteins 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 230000001131 transforming effect Effects 0.000 description 6
- 102100022548 Beta-hexosaminidase subunit alpha Human genes 0.000 description 5
- 101001045440 Homo sapiens Beta-hexosaminidase subunit alpha Proteins 0.000 description 5
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 description 5
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 5
- 102000015098 Tumor Suppressor Protein p53 Human genes 0.000 description 5
- 108010078814 Tumor Suppressor Protein p53 Proteins 0.000 description 5
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 101150113423 hisD gene Proteins 0.000 description 5
- 101150116440 pyrF gene Proteins 0.000 description 5
- 230000035484 reaction time Effects 0.000 description 5
- 239000001044 red dye Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- IBSREHMXUMOFBB-JFUDTMANSA-N 5u8924t11h Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](OC)C[C@H](O[C@@H]2C(=C/C[C@@H]3C[C@@H](C[C@@]4(O3)C=C[C@H](C)[C@@H](C(C)C)O4)OC(=O)[C@@H]3C=C(C)[C@@H](O)[C@H]4OC\C([C@@]34O)=C/C=C/[C@@H]2C)/C)O[C@H]1C.C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 IBSREHMXUMOFBB-JFUDTMANSA-N 0.000 description 4
- 239000005660 Abamectin Substances 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 241000282326 Felis catus Species 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 102000004389 Ribonucleoproteins Human genes 0.000 description 4
- 108010081734 Ribonucleoproteins Proteins 0.000 description 4
- 108020004682 Single-Stranded DNA Proteins 0.000 description 4
- 229950008167 abamectin Drugs 0.000 description 4
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 4
- 229960003669 carbenicillin Drugs 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 230000005782 double-strand break Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000178343 Butea superba Species 0.000 description 3
- 238000010453 CRISPR/Cas method Methods 0.000 description 3
- 101000924727 Homo sapiens Alternative prion protein Proteins 0.000 description 3
- 101000573901 Homo sapiens Major prion protein Proteins 0.000 description 3
- 102100025818 Major prion protein Human genes 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 101150030763 Vegfa gene Proteins 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- 238000012165 high-throughput sequencing Methods 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000008263 repair mechanism Effects 0.000 description 3
- 238000004088 simulation Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 238000007702 DNA assembly Methods 0.000 description 2
- 230000033616 DNA repair Effects 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- 102000005650 Notch Receptors Human genes 0.000 description 2
- 108010070047 Notch Receptors Proteins 0.000 description 2
- 230000006819 RNA synthesis Effects 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 108091027544 Subgenomic mRNA Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 101150063416 add gene Proteins 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 229940104302 cytosine Drugs 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 238000002205 phenol-chloroform extraction Methods 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 108010040467 CRISPR-Associated Proteins Proteins 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 230000008265 DNA repair mechanism Effects 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108010074124 Escherichia coli Proteins Proteins 0.000 description 1
- 239000004258 Ethoxyquin Substances 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000009869 Neu-Laxova syndrome Diseases 0.000 description 1
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- 238000001792 White test Methods 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000007799 cork Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000031877 prophase Effects 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 235000002020 sage Nutrition 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 108091006106 transcriptional activators Proteins 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
提供了一种对细胞基因组中的至少一个靶序列进行基因编辑的基因组编辑系统及方法。该基因组编辑系统含有：(1)包含Cas12f核酸酶的表达构建体；(2)包含与该Cas12f核酸酶相应的引导RNA的表达DNA序列，以及该靶序列的靶向序列的表达构建体。该基因编辑系统或者方法可以将细胞内的目标基因精准敲除；体外切割实验中，能将目标基因精准切断。
Description
PCT国内申请，说明书已公开。
Claims (10)
- PCT国内申请，权利要求书已公开。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2020092799 | 2020-05-28 | ||
| CNPCT/CN2020/092799 | 2020-05-28 | ||
| PCT/CN2020/132759 WO2021238128A1 (zh) | 2020-05-28 | 2020-11-30 | 一种基因组编辑系统及方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115768886A true CN115768886A (zh) | 2023-03-07 |
Family
ID=78745549
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202080101503.1A Pending CN115768886A (zh) | 2020-05-28 | 2020-11-30 | 一种基因组编辑系统及方法 |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US20230212612A1 (zh) |
| EP (1) | EP4159853A4 (zh) |
| CN (1) | CN115768886A (zh) |
| WO (1) | WO2021238128A1 (zh) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116987693A (zh) * | 2022-04-25 | 2023-11-03 | 上海科技大学 | 一种优化的CRISPR/SpCas12f1系统、工程化向导RNA及其应用 |
| CN116987686A (zh) * | 2022-04-25 | 2023-11-03 | 上海科技大学 | 一种工程优化的核酸酶、向导rna、编辑系统和应用 |
| WO2024038168A1 (en) * | 2022-08-19 | 2024-02-22 | UCB Biopharma SRL | Novel rna-guided nucleases and nucleic acid targeting systems comprising such |
| CN117701561A (zh) * | 2022-09-15 | 2024-03-15 | 上海科技大学 | 一种优化的向导RNA、CRISPR/AcC2C9基因编辑系统及基因编辑方法 |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404036B (zh) * | 2014-11-03 | 2017-12-01 | 赛业（苏州）生物科技有限公司 | 基于CRISPR/Cas9技术的条件性基因敲除方法 |
| US10316324B2 (en) * | 2017-08-09 | 2019-06-11 | Benson Hill Biosystems, Inc. | Compositions and methods for modifying genomes |
| WO2019089808A1 (en) * | 2017-11-01 | 2019-05-09 | The Regents Of The University Of California | Class 2 crispr/cas compositions and methods of use |
| CN113136375B (zh) * | 2018-10-29 | 2023-01-06 | 中国农业大学 | 新型CRISPR/Cas12f酶和系统 |
| CN111690717B (zh) * | 2020-04-30 | 2023-05-30 | 山东舜丰生物科技有限公司 | 基于crispr技术进行目标核酸检测的方法和系统 |
| CN111996236B (zh) * | 2020-05-29 | 2021-06-29 | 山东舜丰生物科技有限公司 | 基于crispr技术进行靶核酸检测的方法 |
| CN111690773B (zh) * | 2020-06-17 | 2021-08-20 | 山东舜丰生物科技有限公司 | 利用新型Cas酶进行目标核酸检测的方法和系统 |
| CN111733216B (zh) * | 2020-06-22 | 2023-03-28 | 山东舜丰生物科技有限公司 | 一种提高目标核酸检测效率的方法 |
-
2020
- 2020-11-30 CN CN202080101503.1A patent/CN115768886A/zh active Pending
- 2020-11-30 US US17/927,563 patent/US20230212612A1/en active Pending
- 2020-11-30 WO PCT/CN2020/132759 patent/WO2021238128A1/zh unknown
- 2020-11-30 EP EP20938166.4A patent/EP4159853A4/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| EP4159853A4 (en) | 2023-11-15 |
| WO2021238128A1 (zh) | 2021-12-02 |
| EP4159853A1 (en) | 2023-04-05 |
| US20230212612A1 (en) | 2023-07-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2020267249B2 (en) | Genome editing using campylobacter jejuni crispr/cas system-derived rgen | |
| RU2713328C2 (ru) | Гибридные днк/рнк-полинуклеотиды crispr и способы применения | |
| US12123014B2 (en) | Class II, type V CRISPR systems | |
| US10982200B2 (en) | Enzymes with RuvC domains | |
| CN115768886A (zh) | 一种基因组编辑系统及方法 | |
| CN116694603A (zh) | 新型的Cas蛋白、Crispr-Cas系统及其在基因编辑领域中的用途 | |
| WO2023098485A1 (zh) | 一种基于c2c9核酸酶的新型基因组编辑系统及其应用 | |
| WO2023206871A1 (zh) | 一种优化的CRISPR/SpCas12f1系统、工程化向导RNA及其应用 | |
| US20240200047A1 (en) | Enzymes with ruvc domains | |
| WO2022066335A1 (en) | Systems and methods for transposing cargo nucleotide sequences | |
| EP4200422A1 (en) | Systems and methods for transposing cargo nucleotide sequences | |
| US20130203121A1 (en) | Methods for the semi-synthetic production of high purity "minicircle" dna vectors from plasmids | |
| WO2023206872A1 (zh) | 一种工程优化的核酸酶、向导rna、编辑系统和应用 | |
| WO2024055664A1 (zh) | 一种优化的向导RNA、CRISPR/AcC2C9基因编辑系统及基因编辑方法 | |
| JP2024543186A (ja) | Ｃ２ｃ９ヌクレアーゼに基づく新規なゲノム編集システムおよびその使用 | |
| CN118773168A (zh) | CRISPR/Cas效应蛋白及系统 | |
| CN118207185A (zh) | 一种碱基编辑系统及碱基编辑方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |