|
[[File:GRNA-Cas9.png|thumb|CRISPR/Cas9]]
The CRISPR/Cas system is a [[prokaryotic]] [[immune system]] that confers resistance to foreign genetic elements such as those present within [[Plasmid|plasmids]] and [[phage]]s<ref>{{cite journal | vauthors = Redman M, King A, Watson C, King D | title = What is CRISPR/Cas9? | journal = Archives of Disease in Childhood. Education and Practice Edition | volume = 101 | issue = 4 | pages = 213–5 | date = August 2016 | pmid = 27059283 | pmc = 4975809 | doi = 10.1136/archdischild-2016-310459 }}</ref><ref name="pmid17379808">{{cite journal | vauthors = Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P | display-authors = 6 | title = CRISPR provides acquired resistance against viruses in prokaryotes | journal = Science | volume = 315 | issue = 5819 | pages = 1709–12 | date = March 2007 | pmid = 17379808 | pmc = | doi = 10.1126/science.1138140 | bibcode = 2007Sci...315.1709B }} {{Registration required}}</ref><ref name="pmid19095942">{{cite journal | vauthors = Marraffini LA, Sontheimer EJ | title = CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA | journal = Science | volume = 322 | issue = 5909 | pages = 1843–5 | date = December 2008 | pmid = 19095942 | pmc = 2695655 | doi = 10.1126/science.1165771 | bibcode = 2008Sci...322.1843M }}</ref> that provides a form of [[acquired immunity]]. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA.<ref name=Mohanraju2016>{{cite journal |vauthors = Mohanraju P, Makarova KS, Zetsche B, Zhang F, Koonin EV, van der Oost J |title=Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems |journal=Science |volume=353 |issue=6299 |pages=aad5147 |year=2016 |pmid=27493190 |doi=10.1126/science.aad5147 }}</ref> CRISPR are found in approximately 50% of sequenced [[bacterial genome]]s and nearly 90% of sequenced [[archaea]].<ref name=Hille2018>{{cite journal |vauthors=Hille F, Richter H, Wong SP, Bratovič M, Ressel S, Charpentier E |title=The Biology of CRISPR-Cas: Backward and Forward |journal=Cell |volume=172 |issue=6 |pages=1239–1259 |date=March 2018 |pmid=29522745 |doi=10.1016/j.cell.2017.11.032 }}</ref>
==Use for gene editing==
[[File:Crispr.png|thumb|upright=1.5|right|Diagram of the CRISPR prokaryotic antiviral defense mechanism.<ref name="pmid20056882" />]]
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the [[Cas9]] nuclease complexed with a synthetic [[guide RNA]] (gRNA) into a cell, the cell's [[genome]] can be cut at a desired location, allowing existing genes to be removed and/or new ones added.<ref name="nature99" /><ref name="vb99" /><ref name="pmid26121415">{{cite journal | vauthors = Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB, Bacchetta R, Tsalenko A, Dellinger D, Bruhn L, Porteus MH | title = Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells | journal = Nature Biotechnology | volume = 33 | issue = 9 | pages = 985–9 | date = September 2015 | pmid = 26121415 | doi = 10.1038/nbt.3290 | pmc=4729442}}</ref> The Cas9-gRNA complex corresponds with the CAS III CRISPR-RNA complex in the accompanying diagram.
CRISPR/Cas genome editing techniques have many potential applications, including medicine and crop seed enhancement. The use of CRISPR/Cas9-gRNA complex for [[genome editing]]<ref>{{cite journal | vauthors = Ledford H | title = CRISPR: gene editing is just the beginning | journal = Nature | volume = 531 | issue = 7593 | pages = 156–9 | date = March 2016 | pmid = 26961639 | doi = 10.1038/531156a | bibcode = 2016Natur.531..156L }}</ref><ref>{{Cite web|url=https://www.wired.com/2015/07/crispr-dna-editing-2/|title=The Genesis Engine | last = Maxmen | first = Amy | name-list-format = vanc | date = August 2015 | website = WIRED | access-date = 2016-06-05 }}</ref> was the [[American Association for the Advancement of Science|AAAS]]'s choice for breakthrough of the year in 2015.<ref>{{cite web | vauthors = Travis J | title = Breakthrough of the Year: CRISPR makes the cut | url = http://www.sciencemag.org/news/2015/12/and-science-s-breakthrough-year | website = Science Magazine | publisher = American Association for the Advancement of Science | date = 17 December 2015 }}</ref> [[Bioethical]] concerns have been raised about the prospect of using CRISPR for [[germline]] editing.<ref name="NatNews2015"/>
== History ==
== Applications ==
{{main|CRISPR gene editing}}
By the end of 2014 some 1000 research papers had been published that mentioned CRISPR.<ref>{{cite journal | vauthors = Doudna JA, Charpentier E | title = Genome editing. The new frontier of genome engineering with CRISPR-Cas9 | journal = Science | volume = 346 | issue = 6213 | pages = 1258096 | date = November 2014 | pmid = 25430774 | doi = 10.1126/science.1258096 }}</ref><ref name="NatNews2015">{{cite journal | vauthors = Ledford H | title = CRISPR, the disruptor | journal = Nature | volume = 522 | issue = 7554 | pages = 20–4 | date = June 2015 | pmid = 26040877 | doi = 10.1038/522020a | bibcode = 2015Natur.522...20L }}</ref> The technology had been used to functionally inactivate genes in human cell lines and cells, to study ''[[Candida albicans]]'', to modify [[yeasts]] used to make [[biofuels]] and to [[genetically modified crops|genetically modify crop]] strains.<ref name="NatNews2015" /> CRISPR can also be used to change mosquitos so they cannot transmit diseases such as malaria.<ref name="pmid26849518">{{cite journal | vauthors = Alphey L | title = Can CRISPR-Cas9 gene drives curb malaria? | journal = Nature Biotechnology | volume = 34 | issue = 2 | pages = 149–50 | year = 2016 | pmid = 26849518 | doi = 10.1038/nbt.3473 }}</ref>
[[File:DNA Repair.png|thumb|upright=1.5|DNA repair after double-strand break]]
=== Predecessors ===
In the early 2000s, researchers developed [[zinc finger nuclease]]s (ZFNs), synthetic proteins whose [[DNA-binding domains]] enable them to create double-stranded breaks in DNA at specific points. In 2010, synthetic nucleases called [[transcription activator-like effector nuclease]]s (TALENs) provided an easier way to target a double-stranded break to a specific location on the DNA strand. Both zinc finger nucleases and TALENs require the creation of a custom protein for each targeted DNA sequence, which is a more difficult and time-consuming process than that for guide RNAs. CRISPRs are much easier to design because the process requires making only a short RNA sequence.<ref name="MIT">{{cite journal|last=Young|first=Susan|date=11 February 2014|title=CRISPR and Other Genome Editing Tools Boost Medical Research and Gene Therapy's Reach|url=http://www.technologyreview.com/review/524451/genome-surgery|accessdate=2014-04-13|name-list-format=vanc|journal=[[MIT Technology Review]]}}</ref>
Whereas [[RNA interference|RNA interference (RNAi)]] does not fully suppress gene function, CRISPR, [[Zinc finger nuclease|ZFNs]] and [[Transcription activator-like effector nuclease|TALENs]] provide full irreversible [[gene knockout]].<ref name="Heidenreich_2016">{{cite journal | vauthors = Heidenreich M, Zhang F | title = Applications of CRISPR-Cas systems in neuroscience | journal = Nature Reviews. Neuroscience | volume = 17 | issue = 1 | pages = 36–44 | date = January 2016 | pmid = 26656253 | pmc = 4899966 | doi = 10.1038/nrn.2015.2 }}</ref> CRISPR can also target several DNA sites simultaneously by simply introducing different gRNAs. In addition, CRISPR costs are relatively low.<ref name="Heidenreich_2016" /><ref>{{cite journal | vauthors = Barrangou R, Doudna JA | title = Applications of CRISPR technologies in research and beyond | journal = Nature Biotechnology | volume = 34 | issue = 9 | pages = 933–941 | date = September 2016 | pmid = 27606440 | doi = 10.1038/nbt.3659 }}</ref><ref>{{cite journal | vauthors = Cox DB, Platt RJ, Zhang F | title = Therapeutic genome editing: prospects and challenges | journal = Nature Medicine | volume = 21 | issue = 2 | pages = 121–31 | date = February 2015 | pmid = 25654603 | pmc = 4492683 | doi = 10.1038/nm.3793 }}</ref>
=== Genome engineering ===
CRISPR/Cas9 genome editing is carried out with a [[CRISPR#Cas genes and CRISPR subtypes|Type II]] CRISPR system. When utilized for genome editing, this system includes [[Cas9]], crRNA, tracrRNA along with an optional section of DNA repair template that is utilized in either non-homologous end joining (NHEJ) or homology directed repair (HDR).
[[File:CRISPR overview - en.svg|thumb|upright=1.5|Overview of CRISPR Cas9 plasmid construction<ref>{{cite web | title = CRISPR/Cas9 Plasmids | url = https://www.systembio.com/crispr-cas9/overview | website = www.systembio.com | accessdate = 2015-12-17 }}</ref><ref>{{cite web | title = CRISPR Cas9 Genome Editing | publisher = OriGene | url = http://www.origene.com/crispr-cas9/ | website = www.origene.com | accessdate = 2015-12-17 }}</ref>]]
==== Major components ====
{| class="wikitable"
|-
! Component
! Function
|-
| [[List of RNAs|crRNA]]
| Contains the guide RNA that locates the correct section of host DNA along with a region that binds to [[Trans-activating crRNA|tracrRNA]] (generally in a [[Stem-loop|hairpin loop]] form) forming an active complex.
|-
| [[Trans-activating crRNA|tracrRNA]]
| Binds to [[List of RNAs|crRNA]] and forms an active complex.
|-
| sgRNA
| Single guide RNAs are a combined RNA consisting of a [[Trans-activating crRNA|tracrRNA]] and at least one [[List of RNAs|crRNA]]
|-
| Cas9
| Protein whose active form is able to modify DNA. Many variants exist with differing functions (i.e. single strand nicking, double strand break, DNA binding) due to Cas9's DNA site recognition function.
|-
| Repair template
| DNA that guides the cellular repair process allowing insertion of a specific DNA sequence
|}
CRISPR/Cas9 often employs a [[plasmid]] to [[Transfection|transfect]] the target cells.<ref name="Ran_2013" /> The main components of this plasmid are displayed in the image and listed in the table. The crRNA needs to be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the cell's DNA. The crRNA must bind only where editing is desired. The repair template is designed for each application, as it must overlap with the sequences on either side of the cut and code for the insertion sequence.
Multiple crRNAs and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA).<ref>{{cite thesis |url=https://archive.org/details/LyJosephP201311PhDThesis |title=Discovering Genes Responsible for Kidney Diseases |last=Ly |first=Joseph |year=2013 |type=Ph.D. |publisher=University of Toronto |accessdate=26 December 2016}}</ref> This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells.
[[File:CRISPR transfection.png|thumb|upright=1.5|Overview of the transfection and DNA cleaving by CRISPR Cas9 (crRNA and tracrRNA are often joined as one strand of RNA when designing a plasmid)<ref name="Ran_2013" />]]
==== Structure ====
CRISPR/Cas9 offers a high degree of fidelity and relatively simple construction. It depends on two factors for its specificity: the target sequence and the PAM. The target sequence is 20 bases long as part of each CRISPR locus in the crRNA array.<ref name="Ran_2013">{{cite journal | vauthors = Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F | title = Genome engineering using the CRISPR-Cas9 system | journal = Nature Protocols | volume = 8 | issue = 11 | pages = 2281–308 | date = November 2013 | pmid = 24157548 | pmc = 3969860 | doi = 10.1038/nprot.2013.143 | hdl = 1721.1/102943 }}</ref> A typical crRNA array has multiple unique target sequences. Cas9 proteins select the correct location on the host's genome by utilizing the sequence to bond with base pairs on the host DNA. The sequence is not part of the Cas9 protein and as a result is customizable and can be independently [[Oligonucleotide synthesis|synthesized]].<ref>{{cite journal | vauthors = Horvath P, Barrangou R | title = CRISPR/Cas, the immune system of bacteria and archaea | journal = Science | volume = 327 | issue = 5962 | pages = 167–70 | date = January 2010 | pmid = 20056882 | doi = 10.1126/science.1179555 | bibcode = 2010Sci...327..167H }}</ref><ref>{{cite journal | vauthors = Bialk P, Rivera-Torres N, Strouse B, Kmiec EB | title = Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems | journal = PLOS One | volume = 10 | issue = 6 | pages = e0129308 | date = 2015-06-08 | pmid = 26053390 | pmc = 4459703 | doi = 10.1371/journal.pone.0129308 | bibcode = 2015PLoSO..1029308B }}</ref>
The PAM sequence on the host genome is recognized by Cas9. Cas9 cannot be easily modified to recognize a different PAM sequence. However this is not too limiting as it is a short sequence and nonspecific (e.g. the SpCas9 PAM sequence is 5'-NGG-3' and in the human genome occurs roughly every 8 to 12 base pairs).<ref name="Ran_2013" />
Once these have been assembled into a plasmid and transfected into cells the Cas9 protein with the help of the crRNA finds the correct sequence in the host cell's DNA and – depending on the Cas9 variant – creates a single or double strand break in the DNA.<ref>{{cite web |url=http://news.berkeley.edu/2015/11/12/crispr-cas9-gene-editing-check-three-times-cut-once/ |title=CRISPR-Cas9 gene editing: check three times, cut once | first = Robert | last = Sanders | name-list-format = vanc |date=12 November 2015 |publisher=University of California, Berkeley |accessdate=26 December 2016 |archiveurl=https://web.archive.org/web/20161226231842/http://news.berkeley.edu/2015/11/12/crispr-cas9-gene-editing-check-three-times-cut-once/ |archivedate=26 December 2016 |deadurl=no}}</ref>
Properly spaced single strand breaks in the host DNA can trigger [[homology directed repair]], which is less error prone than the non-homologous end joining that typically follows a double strand break. Providing a DNA repair template allows for the insertion of a specific DNA sequence at an exact location within the genome. The repair template should extend 40 to 90 base pairs beyond the Cas9 induced DNA break.<ref name="Ran_2013" /> The goal is for the cell's HDR process to utilize the provided repair template and thereby incorporate the new sequence into the genome. Once incorporated, this new sequence is now part of the cell's genetic material and passes into its daughter cells.
Many online tools are available to aid in designing effective sgRNA sequences.<ref>{{cite web|title = Optimized CRISPR Design|url = http://crispr.mit.edu/|website = crispr.mit.edu|accessdate = 2015-12-20}}</ref>
==== Delivery ====
{{See also|Transfection}}
Delivery of Cas9, sgRNA, and associated complexes into cells can occur via viral and non-viral systems. [[Electroporation]] of DNA, RNA, or ribonucleocomplexes is a common technique, though it can result in harmful effects on the target cells.<ref name="Lino_2018">{{cite journal | vauthors = Lino CA, Harper JC, Carney JP, Timlin JA | title = Delivering CRISPR: a review of the challenges and approaches | journal = Drug Delivery | volume = 25 | issue = 1 | pages = 1234–1257 | date = November 2018 | pmid = 29801422 | pmc = 6058482 | doi = 10.1080/10717544.2018.1474964 }}</ref> Chemical transfection techniques utilizing [[lipid]]s have also been used to introduce sgRNA in complex with Cas9 into cells.<ref name="Li_2018">{{cite journal | vauthors = Li L, Hu S, Chen X | title = Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities | journal = Biomaterials | volume = 171 | issue = | pages = 207–218 | date = July 2018 | pmid = 29704747 | pmc = 5944364 | doi = 10.1016/j.biomaterials.2018.04.031 }}</ref> Hard-to-transfect cells (e.g. stem cells, neurons, and hematopoietic cells) require more efficient delivery systems such as those based on [[lentivirus]] (LVs), [[Adenoviridae|adenovirus]] (AdV) and [[adeno-associated virus]] (AAV).<ref name="Schmidt_2015">{{cite journal | vauthors = Schmidt F, Grimm D | title = CRISPR genome engineering and viral gene delivery: a case of mutual attraction | journal = Biotechnology Journal | volume = 10 | issue = 2 | pages = 258–72 | date = February 2015 | pmid = 25663455 | doi = 10.1002/biot.201400529 }}</ref><ref>{{cite web| title = CRISPR 101: Mammalian Expression Systems and Delivery Methods | first1 = Nicole | last1 = Waxmonsky | name-list-format = vanc | url = https://blog.addgene.org/crispr-101-mammalian-expression-systems-and-delivery-methods | date = 24 September 2015 | access-date = 11 June 2018 }}</ref>
==== Editing ====
CRISPRs have been used to cut five<ref name=craze/> to 62 genes at once: pig cells have been engineered to inactivate all 62 [[Porcine endogenous retroviruses|Porcine Endogenous Retroviruses]] in the pig genome, which eliminated transinfection from the pig to human cells in culture.<ref>{{cite news | newspaper=NY Times | url=https://www.nytimes.com/2015/10/20/science/editing-of-pig-dna-may-lead-to-more-organs-for-people.html?_r=1 | first = Carl | last = Zimmerman | name-list-format = vanc | title=Editing of Pig DNA May Lead to More Organs for People | date=Oct 15, 2015}}</ref> CRISPR's low cost compared to alternatives is widely seen as revolutionary.<ref name="nature99">{{cite journal |doi=10.1038/522020a |pmid=26040877 |title=CRISPR, the disruptor |journal=Nature |volume=522 |issue=7554 |pages=20–4 |year=2015 |last1=Ledford |first1=Heidi | name-list-format = vanc | bibcode=2015Natur.522...20L }}</ref><ref name="vb99">{{cite web|url = http://news.vanderbilt.edu/2014/08/new-technique-accelerates-genome-editing-process/|title = New technique accelerates genome editing process|date = 21 August 2014|accessdate = |website = research news @ Vanderbilt|publisher = Vanderbilt University|last = Snyder|first = Bill | name-list-format = vanc | location = Nashville, Tennessee}}</ref>
Selective engineered redirection of the CRISPR/Cas system was first demonstrated in 2012 in:<ref name=halemajumder2012>{{cite journal | vauthors = Hale CR, Majumdar S, Elmore J, Pfister N, Compton M, Olson S, Resch AM, Glover CV, Graveley BR, Terns RM, Terns MP | title = Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs | journal = Molecular Cell | volume = 45 | issue = 3 | pages = 292–302 | date = February 2012 | pmid = 22227116 | pmc = 3278580 | doi = 10.1016/j.molcel.2011.10.023 }}</ref><ref>{{cite journal | vauthors = Sorek R, Kunin V, Hugenholtz P | title = CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea | journal = Nature Reviews. Microbiology | volume = 6 | issue = 3 | pages = 181–6 | date = March 2008 | pmid = 18157154 | doi = 10.1038/nrmicro1793 }}</ref>
* Immunization of industrially important bacteria, including some used in food production and large-scale fermentation
* Cellular or organism RNA-guided [[genome engineering]]. Proof of concept studies demonstrated examples both ''[[in vitro]]''<ref name=pmid26121415/><ref name="pmid22745249">{{cite journal | vauthors = Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E | title = A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity | journal = Science | volume = 337 | issue = 6096 | pages = 816–21 | date = August 2012 | pmid = 22745249 | pmc = | doi = 10.1126/science.1225829 | bibcode = 2012Sci...337..816J }}</ref><ref name="pmid22949671"/> and ''[[in vivo]]''<ref name="pmid23643243"/><ref name="Cong2013"/><ref>{{cite journal | vauthors = Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM | title = RNA-guided human genome engineering via Cas9 | journal = Science | volume = 339 | issue = 6121 | pages = 823–6 | date = February 2013 | pmid = 23287722 | pmc = 3712628 | doi = 10.1126/science.1232033 | bibcode = 2013Sci...339..823M }}<br/>{{cite journal | vauthors = Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA | title = Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 110 | issue = 39 | pages = 15644–9 | date = September 2013 | pmid = 23940360| pmc = 3785731| doi = 10.1073/pnas.1313587110 | bibcode = 2013PNAS..11015644H }}</ref>
==== Controlled genome editing ====
Several variants of CRISPR/Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules.<ref name=":1">{{cite journal | vauthors = Oakes BL, Nadler DC, Flamholz A, Fellmann C, Staahl BT, Doudna JA, Savage DF | title = Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch | journal = Nature Biotechnology | volume = 34 | issue = 6 | pages = 646–51 | date = June 2016 | pmid = 27136077 | pmc = 4900928 | doi = 10.1038/nbt.3528 }}</ref><ref name="pmid26857072">{{cite journal | vauthors = Nuñez JK, Harrington LB, Doudna JA | title = Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering | journal = ACS Chemical Biology | volume = 11 | issue = 3 | pages = 681–8 | date = March 2016 | pmid = 26857072 | doi = 10.1021/acschembio.5b01019 }}</ref><ref name="pmid26996256">{{cite journal | vauthors = Zhou W, Deiters A | title = Conditional Control of CRISPR/Cas9 Function | journal = Angewandte Chemie | volume = 55 | issue = 18 | pages = 5394–9 | date = April 2016 | pmid = 26996256 | doi = 10.1002/anie.201511441 }}</ref> These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 for gene activation,<ref name="pmid25664691">{{cite journal | vauthors = Polstein LR, Gersbach CA | title = A light-inducible CRISPR-Cas9 system for control of endogenous gene activation | journal = Nature Chemical Biology | volume = 11 | issue = 3 | pages = 198–200 | date = March 2015 | pmid = 25664691 | pmc = 4412021 | doi = 10.1038/nchembio.1753 }}</ref><ref name="pmid25619936">{{cite journal | vauthors = Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M | title = CRISPR-Cas9-based photoactivatable transcription system | journal = Chemistry & Biology | volume = 22 | issue = 2 | pages = 169–74 | date = February 2015 | pmid = 25619936 | doi = 10.1016/j.chembiol.2014.12.011 }}</ref> or fusing similar light responsive domains with two constructs of split-Cas9,<ref name="pmid25713377">{{cite journal | vauthors = Wright AV, Sternberg SH, Taylor DW, Staahl BT, Bardales JA, Kornfeld JE, Doudna JA | title = Rational design of a split-Cas9 enzyme complex | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 112 | issue = 10 | pages = 2984–9 | date = March 2015 | pmid = 25713377 | pmc = 4364227 | doi = 10.1073/pnas.1501698112 | bibcode = 2015PNAS..112.2984W }}</ref><ref name="pmid26076431">{{cite journal | vauthors = Nihongaki Y, Kawano F, Nakajima T, Sato M | title = Photoactivatable CRISPR-Cas9 for optogenetic genome editing | journal = Nature Biotechnology | volume = 33 | issue = 7 | pages = 755–60 | date = July 2015 | pmid = 26076431 | doi = 10.1038/nbt.3245 }}</ref> or by incorporating caged unnatural amino acids into Cas9,<ref name="pmid25905628">{{cite journal | vauthors = Hemphill J, Borchardt EK, Brown K, Asokan A, Deiters A | title = Optical Control of CRISPR/Cas9 Gene Editing | journal = Journal of the American Chemical Society | volume = 137 | issue = 17 | pages = 5642–5 | date = May 2015 | pmid = 25905628 | pmc = 4919123 | doi = 10.1021/ja512664v }}</ref> or by modifying the guide RNAs with photocleavable complements for genome editing.<ref name="pmid27554600">{{cite journal | vauthors = Jain PK, Ramanan V, Schepers AG, Dalvie NS, Panda A, Fleming HE, Bhatia SN | title = Development of Light-Activated CRISPR Using Guide RNAs with Photocleavable Protectors | journal = Angewandte Chemie | volume = 55 | issue = 40 | pages = 12440–4 | date = September 2016 | pmid = 27554600 | pmc = 5864249 | doi = 10.1002/anie.201606123 }}</ref>
Methods to control genome editing with small molecules include an allosteric Cas9, with no detectable background editing, that will activate binding and cleavage upon the addition of [[Afimoxifene|4-hydroxytamoxifen]] (4-HT),<ref name=":1" /> 4-HT responsive [[intein]]-linked Cas9s<ref>{{cite journal | vauthors = Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR | title = Small molecule-triggered Cas9 protein with improved genome-editing specificity | journal = Nature Chemical Biology | volume = 11 | issue = 5 | pages = 316–8 | date = May 2015 | pmid = 25848930 | pmc = 4402137 | doi = 10.1038/nchembio.1793 }}</ref> or a Cas9 that is 4-HT responsive when fused to four ERT2 domains.<ref>{{cite journal | vauthors = Liu KI, Ramli MN, Woo CW, Wang Y, Zhao T, Zhang X, Yim GR, Chong BY, Gowher A, Chua MZ, Jung J, Lee JH, Tan MH | title = A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing | journal = Nature Chemical Biology | volume = 12 | issue = 11 | pages = 980–987 | date = November 2016 | pmid = 27618190 | doi = 10.1038/nchembio.2179 }}</ref> Intein-inducible split-Cas9 allows [[dimerization (chemistry)|dimerization]] of Cas9 fragments<ref>{{cite journal | vauthors = Truong DJ, Kühner K, Kühn R, Werfel S, Engelhardt S, Wurst W, Ortiz O | title = Development of an intein-mediated split-Cas9 system for gene therapy | journal = Nucleic Acids Research | volume = 43 | issue = 13 | pages = 6450–8 | date = July 2015 | pmid = 26082496 | pmc = 4513872 | doi = 10.1093/nar/gkv601 }}</ref> and [[Sirolimus|Rapamycin]]-inducible split-Cas9 system developed by fusing two constructs of split Cas9 with FRB and [[FKBP]] fragments.<ref>{{cite journal | vauthors = Zetsche B, Volz SE, Zhang F | title = A split-Cas9 architecture for inducible genome editing and transcription modulation | journal = Nature Biotechnology | volume = 33 | issue = 2 | pages = 139–42 | date = February 2015 | pmid = 25643054 | pmc = 4503468 | doi = 10.1038/nbt.3149 }}</ref> Furthermore, other studies have shown to induce transcription of Cas9 with a small molecule, [[doxycycline]].<ref>{{cite journal | vauthors = González F, Zhu Z, Shi ZD, Lelli K, Verma N, Li QV, Huangfu D | title = An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells | journal = Cell Stem Cell | volume = 15 | issue = 2 | pages = 215–26 | date = August 2014 | pmid = 24931489 | pmc = 4127112 | doi = 10.1016/j.stem.2014.05.018 }}</ref><ref>{{cite journal | vauthors = Dow LE, Fisher J, O'Rourke KP, Muley A, Kastenhuber ER, Livshits G, Tschaharganeh DF, Socci ND, Lowe SW | title = Inducible in vivo genome editing with CRISPR-Cas9 | journal = Nature Biotechnology | volume = 33 | issue = 4 | pages = 390–4 | date = April 2015 | pmid = 25690852 | pmc = 4390466 | doi = 10.1038/nbt.3155 }}</ref> Small molecules can also be used to improve Homology Directed Repair (HDR),<ref>{{cite journal | vauthors = Yu C, Liu Y, Ma T, Liu K, Xu S, Zhang Y, Liu H, La Russa M, Xie M, Ding S, Qi LS | title = Small molecules enhance CRISPR genome editing in pluripotent stem cells | journal = Cell Stem Cell | volume = 16 | issue = 2 | pages = 142–7 | date = February 2015 | pmid = 25658371 | pmc = 4461869 | doi = 10.1016/j.stem.2015.01.003 }}</ref> often by inhibiting the Non-Homologous End Joining (NHEJ) pathway.<ref>{{cite journal | vauthors = Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL | title = Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining | journal = Nature Biotechnology | volume = 33 | issue = 5 | pages = 538–42 | date = May 2015 | pmid = 25798939 | pmc = 4618510 | doi = 10.1038/nbt.3190 }}</ref> These systems allow conditional control of CRISPR activity for improved precision, efficiency and spatiotemporal control.
=== Knockdown/activation ===
{{main|CRISPR interference}}
[[File:Dead-Cas9 potential applications.png|thumb|upright=1.5|A dead Cas9 protein coupled with epigenetic modifiers which are used to repress certain genome sequences rather than cutting it all together.<ref>{{Cite journal|last=Ledford|first=Heidi|date=2016-03-07|title=CRISPR: gene editing is just the beginning|url=https://www.nature.com/news/crispr-gene-editing-is-just-the-beginning-1.19510|journal=Nature|language=en|volume=531|issue=7593|pages=156–159|doi=10.1038/531156a|pmid=26961639|issn=0028-0836|bibcode=2016Natur.531..156L}}</ref>]]
Using "dead" versions of Cas9 ([[Cas9#Interference of transcription by dCas9|dCas9]]) eliminates CRISPR's DNA-cutting ability, while preserving its ability to target desirable sequences. Multiple groups added various regulatory factors to dCas9s, enabling them to turn almost any gene on or off or adjust its level of activity.<ref name="Science_Breakthrough">{{cite web | title = And Science's Breakthrough of the Year is …|url = http://news.sciencemag.org/scientific-community/2015/12/and-science-s-breakthrough-year|website = news.sciencemag.org|accessdate = 2015-12-21|date = December 17, 2015|last = Science News Staff}}</ref> Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. The targeted site is methylated, [[Epigenetics|epigenetically]] modifying the gene. This modification inhibits transcription. These precisely placed modifications may then be used to regulate the effects on gene expressions and DNA dynamics after the inhibition of certain genome sequences within DNA. Within the past few years, epigenetic marks in different human cells have been closely researched and certain patterns within the marks have been found to correlate with everything ranging from tumor growth to brain activity.<ref>{{cite journal | vauthors = Ledford H | title = CRISPR: gene editing is just the beginning | journal = Nature | volume = 531 | issue = 7593 | pages = 156–9 | date = March 2016 | pmid = 26961639 | doi = 10.1038/531156a | url = https://www.nature.com/news/crispr-gene-editing-is-just-the-beginning-1.19510 | bibcode = 2016Natur.531..156L }}</ref> Conversely, CRISPR-mediated activation (CRISPRa) promotes gene transcription.<ref>{{cite journal | vauthors = Dominguez AA, Lim WA, Qi LS | title = Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation | journal = Nature Reviews Molecular Cell Biology | volume = 17 | issue = 1 | pages = 5–15 | date = January 2016 | pmid = 26670017 | pmc = 4922510 | doi = 10.1038/nrm.2015.2 }}</ref> Cas9 is an effective way of targeting and silencing specific genes at the DNA level.<ref name="pmid24336571">{{cite journal | vauthors = Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F | title = Genome-scale CRISPR-Cas9 knockout screening in human cells | journal = Science | volume = 343 | issue = 6166 | pages = 84–7 | date = January 2014 | pmid = 24336571 | pmc = 4089965 | doi = 10.1126/science.1247005 | bibcode = 2014Sci...343...84S }}</ref> In bacteria, the presence of Cas9 alone is enough to block transcription. For mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA sequences called [[Promoter (genetics)|promoters]] that immediately precede the target gene.<ref name=craze/>
Cas9 was used to carry synthetic [[transcription factor]]s that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter.<ref name=craze/>
=== RNA editing ===
In 2016, researchers demonstrated that CRISPR from an ordinary mouth bacterium could be used to edit [[RNA]]. The researchers searched databases containing hundreds of millions of genetic sequences for those that resembled Crispr genes. They considered the [[fusobacteria]] ''Leptotrichia shahii''. It had a group of genes that resembled CRISPR genes, but with important differences. When the researchers equipped other bacteria with these genes, which they called C2c2, they found that the organisms gained a novel defense.<ref name="Zimmer_2016">{{cite news | url = https://www.nytimes.com/2016/06/04/science/rna-c2c2-gene-editing-dna-crispr.html | title = Scientists Find Form of Crispr Gene Editing With New Capabilities|last=Zimmer|first=Carl | name-list-format = vanc | date=2016-06-03|newspaper=The New York Times|issn=0362-4331|access-date=2016-06-10}}</ref>
Many viruses encode their genetic information in RNA rather than DNA that they repurpose to make new viruses. [[HIV]] and [[poliovirus]] are such viruses. Bacteria with C2c2 make molecules that can dismember RNA, destroying the virus. Tailoring these genes opened any RNA molecule to editing.<ref name="Zimmer_2016" />
CRISPR-Cas systems can also be employed for editing of [[MicroRNA|micro-RNA]] and [[Long non-coding RNA|long-noncoding RNA]] genes in plants.<ref>{{cite journal | vauthors = Basak J, Nithin C | title = Targeting Non-Coding RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting | journal = Frontiers in Plant Science | volume = 6 | pages = 1001 | date = 2015 | pmid = 26635829 | pmc = 4652605 | doi = 10.3389/fpls.2015.01001 }}</ref>
=== Disease models ===
CRISPR simplifies creation of [[Genetically modified organism#Mammals|animals for research]] that mimic disease or show what happens when a gene is [[Gene knockdown|knocked down]] or mutated. CRISPR may be used at the [[germline]] level to create animals where the gene is changed everywhere, or it may be targeted at non-germline cells.<ref name="pmid25914022">{{cite journal | vauthors = van Erp PB, Bloomer G, Wilkinson R, Wiedenheft B | title = The history and market impact of CRISPR RNA-guided nucleases | journal = Current Opinion in Virology | volume = 12 | issue = | pages = 85–90 | date = June 2015 | pmid = 25914022 | doi = 10.1016/j.coviro.2015.03.011 | pmc = 4470805}}</ref><ref name="pmid25819765">{{cite journal | vauthors = Maggio I, Gonçalves MA | title = Genome editing at the crossroads of delivery, specificity, and fidelity | journal = Trends in Biotechnology | volume = 33 | issue = 5 | pages = 280–91 | date = May 2015 | pmid = 25819765 | doi = 10.1016/j.tibtech.2015.02.011 }}</ref><ref name="pmid25868999">{{cite journal | vauthors = Rath D, Amlinger L, Rath A, Lundgren M | title = The CRISPR-Cas immune system: biology, mechanisms and applications | journal = Biochimie | volume = 117 | issue = | pages = 119–28 | date = October 2015 | pmid = 25868999 | doi = 10.1016/j.biochi.2015.03.025 }}</ref>
CRISPR can be utilized to create human cellular models of disease. For instance, applied to human [[Cell potency|pluripotent stem cells]] CRISPR introduced targeted mutations in genes relevant to [[polycystic kidney disease]] (PKD) and [[focal segmental glomerulosclerosis]] (FSGS).<ref name="ReferenceA">{{cite journal | vauthors = Freedman BS, Brooks CR, Lam AQ, Fu H, Morizane R, Agrawal V, Saad AF, Li MK, Hughes MR, Werff RV, Peters DT, Lu J, Baccei A, Siedlecki AM, Valerius MT, Musunuru K, McNagny KM, Steinman TI, Zhou J, Lerou PH, Bonventre JV | display-authors = 6 | title = Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids | journal = Nature Communications | volume = 6 | pages = 8715 | date = October 2015 | pmid = 26493500 | pmc = 4620584 | doi = 10.1038/ncomms9715 | bibcode = 2015NatCo...6E8715F }}</ref> These CRISPR-modified pluripotent stem cells were subsequently grown into human kidney [[organoid]]s that exhibited disease-specific phenotypes. Kidney [[organoid]]s from stem cells with PKD mutations formed large, translucent cyst structures from kidney tubules. The cysts were capable of reaching macroscopic dimensions, up to one centimeter in diameter.<ref>{{cite journal | vauthors = Cruz NM, Song X, Czerniecki SM, Gulieva RE, Churchill AJ, Kim YK, Winston K, Tran LM, Diaz MA, Fu H, Finn LS, Pei Y, Himmelfarb J, Freedman BS | display-authors = 6 | title = Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease | journal = Nature Materials | volume = 16 | issue = 11 | pages = 1112–1119 | date = November 2017 | pmid = 28967916 | pmc = 5936694 | doi = 10.1038/nmat4994 | bibcode = 2017NatMa..16.1112C }}</ref> Kidney organoids with mutations in a gene linked to FSGS developed junctional defects between [[podocyte]]s, the filtering cells affected in that disease. This was traced to the inability of podocytes ability to form microvilli between adjacent cells.<ref>{{cite journal | vauthors = Kim YK, Refaeli I, Brooks CR, Jing P, Gulieva RE, Hughes MR, Cruz NM, Liu Y, Churchill AJ, Wang Y, Fu H, Pippin JW, Lin LY, Shankland SJ, Vogl AW, McNagny KM, Freedman BS | display-authors = 6 | title = Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development | journal = Stem Cells | volume = 35 | issue = 12 | pages = 2366–2378 | date = December 2017 | pmid = 28905451 | doi = 10.1002/stem.2707 | pmc=5742857}}</ref> Importantly, these disease phenotypes were absent in control organoids of identical genetic background, but lacking the CRISPR modifications.<ref name="ReferenceA"/>
A similar approach was taken to model long QT syndrome in [[Cardiac muscle cell|cardiomyocytes]] derived from pluripotent stem cells.<ref>{{cite journal | vauthors = Bellin M, Casini S, Davis RP, D'Aniello C, Haas J, Ward-van Oostwaard D, Tertoolen LG, Jung CB, Elliott DA, Welling A, Laugwitz KL, Moretti A, Mummery CL | title = Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome | journal = The EMBO Journal | volume = 32 | issue = 24 | pages = 3161–75 | date = December 2013 | pmid = 24213244 | pmc = 3981141 | doi = 10.1038/emboj.2013.240 }}</ref> These CRISPR-generated cellular models, with isogenic controls, provide a new way to study human disease and test drugs.
=== Gene drive ===
{{Main|Gene drive}}
Gene drives may provide a powerful tool to restore balance of ecosystems by eliminating invasive species. Concerns regarding efficacy, unintended consequences in the target species as well as non-target species have been raised particularly in the potential for accidental release from laboratories into the wild. Scientists have proposed several safeguards for ensuring the containment of experimental gene drives including molecular, reproductive, and ecological.<ref>{{cite journal | vauthors = Akbari OS, Bellen HJ, Bier E, Bullock SL, Burt A, Church GM, Cook KR, Duchek P, Edwards OR, Esvelt KM, Gantz VM, Golic KG, Gratz SJ, Harrison MM, Hayes KR, James AA, Kaufman TC, Knoblich J, Malik HS, Matthews KA, O'Connor-Giles KM, Parks AL, Perrimon N, Port F, Russell S, Ueda R, Wildonger J | display-authors = 6 | title = BIOSAFETY. Safeguarding gene drive experiments in the laboratory | journal = Science | volume = 349 | issue = 6251 | pages = 927–9 | date = August 2015 | pmid = 26229113 | pmc = 4692367 | doi = 10.1126/science.aac7932 | bibcode = 2015Sci...349..927A }}</ref> Many recommend that immunization and reversal drives be developed in tandem with gene drives in order to overwrite their effects if necessary.<ref>{{cite journal | vauthors = Caplan AL, Parent B, Shen M, Plunkett C | title = No time to waste--the ethical challenges created by CRISPR: CRISPR/Cas, being an efficient, simple, and cheap technology to edit the genome of any organism, raises many ethical and regulatory issues beyond the use to manipulate human germ line cells | journal = EMBO Reports | volume = 16 | issue = 11 | pages = 1421–6 | date = November 2015 | pmid = 26450575 | pmc = 4641494 | doi = 10.15252/embr.201541337 }}</ref> There remains consensus that long-term effects must be studied more thoroughly particularly in the potential for ecological disruption that cannot be corrected with reversal drives.<ref>{{cite journal | vauthors = Oye KA, Esvelt K, Appleton E, Catteruccia F, Church G, Kuiken T, Lightfoot SB, McNamara J, Smidler A, Collins JP | display-authors = 6 | title = Biotechnology. Regulating gene drives | journal = Science | volume = 345 | issue = 6197 | pages = 626–8 | date = August 2014 | pmid = 25035410 | doi = 10.1126/science.1254287 | bibcode = 2014Sci...345..626O }}</ref>
=== Biomedicine ===
CRISPR/Cas technology has been proposed as a treatment for multiple human diseases, especially those with a genetic cause.<ref>{{Cite journal |date= December 2016| title=CRISPR-mediated genome editing and human diseases |journal=Genes & Diseases |volume=3|issue=4|pages=244–251|doi=10.1016/j.gendis.2016.07.003 |last1=Cai |first1=Liquan |last2=Fisher |first2=Alfred L |last3=Huang |first3=Haochu |last4=Xie |first4=Zijian | name-list-format = vanc }}</ref> Its ability to modify specific DNA sequences makes it a tool with potential to fix disease-causing mutations. Early research in animal models suggest that therapies based on CRISPR technology have potential to treat a wide range of diseases,<ref>{{Cite news|url=https://labiotech.eu/tops/crispr-technology-cure-disease/|title=Seven Diseases That CRISPR Technology Could Cure|date=2018-06-25|work=Labiotech.eu|access-date=2018-08-22 }}</ref> including cancer,<ref>{{Cite news |last=Rana|first=Preetika|last2=Marcus|first2=Amy Dockser |first3=Wenxin | name-list-format = vanc |last3=Fan|url=https://www.wsj.com/articles/china-unhampered-by-rules-races-ahead-in-gene-editing-trials-1516562360|title=China, Unhampered by Rules, Races Ahead in Gene-Editing Trials|date=2018-01-21|work=Wall Street Journal|access-date=2018-08-22 }}</ref><ref>{{Cite news|url=https://www.technologyreview.com/s/609999/us-doctors-plan-to-treat-cancer-patients-using-crispr/|title=The first human CRISPR study in the U.S. could begin at any time|last=Mullin|first=Emily | name-list-format = vanc |work=MIT Technology Review|access-date=2018-08-22 }}</ref> beta-thalassemia,<ref>{{cite journal | vauthors = Xie F, Ye L, Chang JC, Beyer AI, Wang J, Muench MO, Kan YW | title = Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac | journal = Genome Research | volume = 24 | issue = 9 | pages = 1526–33 | date = September 2014 | pmid = 25096406 | pmc = 4158758 | doi = 10.1101/gr.173427.114 }}</ref> sickle cell disease,<ref>{{Cite journal|last=Ledford|first=Heidi | name-list-format = vanc |date=2016-10-12|title=CRISPR deployed to combat sickle-cell anaemia |journal=Nature |doi=10.1038/nature.2016.20782 }}</ref> hemophilia,<ref>{{Cite web|url=https://www.genengnews.com/gen-news-highlights/crispr-one-shot-cell-therapy-for-hemophilia-developed/81255772|title=CRISPR "One Shot Cell Therapy for Hemophilia Developed {{!}} GEN|website=GEN|access-date=2018-08-22}}</ref> cystic fibrosis,<ref>{{cite journal | vauthors = Marangi M, Pistritto G | title = Innovative Therapeutic Strategies for Cystic Fibrosis: Moving Forward to CRISPR Technique | journal = Frontiers in Pharmacology | volume = 9 | pages = 396 | date = 2018-04-20 | pmid = 29731717 | pmc = 5920621 | doi = 10.3389/fphar.2018.00396 }}</ref> Duchenne's muscular dystrophy,<ref>{{Cite news|url=https://www.sciencedaily.com/releases/2018/02/180206121017.htm|title=New CRISPR method efficiently corrects Duchenne muscular dystrophy defect in heart tissue|work=ScienceDaily|access-date=2018-08-22 }}</ref> Huntington's,<ref>{{cite journal | vauthors = Eisenstein M | title = CRISPR takes on Huntington's disease | journal = Nature | volume = 557 | issue = 7707 | pages = S42–S43 | date = May 2018 | pmid = 29844549 | doi = 10.1038/d41586-018-05177-y }}</ref><ref>{{cite journal | vauthors = Dabrowska M, Juzwa W, Krzyzosiak WJ, Olejniczak M | title = Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases | journal = Frontiers in Neuroscience | volume = 12 | pages = 75 | date = 2018 | pmid = 29535594 | pmc = 5834764 | doi = 10.3389/fnins.2018.00075 }}</ref> and heart disease.<ref>{{cite journal | vauthors = King A | title = A CRISPR edit for heart disease | journal = Nature | volume = 555 | issue = 7695 | pages = S23–S25 | date = March 2018 | pmid = 29517035 | doi = 10.1038/d41586-018-02482-4 }}</ref>
CRISPR/Cas-based "RNA-guided nucleases" can be used to target [[virulence factors]], genes encoding [[antibiotic resistance]] and other medically relevant sequences of interest. This technology thus represents a novel form of antimicrobial therapy and a strategy by which to manipulate bacterial populations.<ref name="Gomaaetal2014">{{cite journal | vauthors = Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, Beisel CL| title = Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems | journal = MBio | volume = 5 | issue = 1 | pages = e00928–13 | date = January 2014 | pmid = 24473129 | pmc = 3903277 | doi = 10.1128/mBio.00928-13 }}</ref><ref name="CitorikMimee2014">{{cite journal | vauthors = Citorik RJ, Mimee M, Lu TK | title = Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases | journal = Nature Biotechnology | volume = 32 | issue = 11 | pages = 1141–5 | date = November 2014 | pmid = 25240928 | pmc = 4237163 | doi = 10.1038/nbt.3011 | hdl = 1721.1/100834 }}</ref> Recent studies suggested a correlation between the interfering of the CRISPR/Cas locus and acquisition of antibiotic resistance<ref name="Gholizadeh2017">{{cite journal | vauthors = Gholizadeh P, Aghazadeh M, Asgharzadeh M, Kafil HS,| title = Suppressing the CRISPR/Cas adaptive immune system in bacterial infections | journal = European Journal of Clinical Microbiology & Infectious Diseases | volume = 36 | issue = 11 | pages = 2043–2051 | date = October 2017 | pmid = 28601970 | doi = 10.1007/s10096-017-3036-2 }}</ref> This system provides protection of bacteria against invading foreign DNA, such as transposons, bacteriophages and plasmids. This system was shown to be a strong selective pressure for the acquisition of antibiotic resistance and virulence factor in bacterial pathogens.<ref name="Gholizadeh2017" /> Some of the affected genes are tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and fetal [[hemoglobin]].<ref name="craze" />
Research suggests that CRISPR is an effective way to limit replication of multiple [[herpesviruses]]. It was able to eradicate viral DNA in the case of [[Epstein-Barr virus]] (EBV). Anti-herpesvirus CRISPRs have promising applications such as removing cancer-causing EBV from tumor cells, helping rid donated organs for [[immunocompromised]] patients of viral invaders, or preventing [[Herpes labialis|cold sore]] outbreaks and recurrent eye infections by blocking [[HSV-1]] reactivation. {{as of|2016|August}}, these were awaiting testing.<ref name="pmid27362483">{{cite journal | vauthors = van Diemen FR, Kruse EM, Hooykaas MJ, Bruggeling CE, Schürch AC, van Ham PM, Imhof SM, Nijhuis M, Wiertz EJ, Lebbink RJ | title = CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections | journal = PLoS Pathogens | volume = 12 | issue = 6 | pages = e1005701 | year = 2016 | pmid = 27362483 | pmc = 4928872 | doi = 10.1371/journal.ppat.1005701 | laysummary = https://www.youtube.com/watch?v=lQaWh8VLkiU | laysource = PLOS Media YouTube Channel }}</ref> CRISPR is being applied to develop tissue-based treatments for cancer and other diseases.<ref name="Science_Breakthrough" /><ref name="pmid27595406">{{cite journal | vauthors = Liu Y, Zhan Y, Chen Z, He A, Li J, Wu H, Liu L, Zhuang C, Lin J, Guo X, Zhang Q, Huang W, Cai Z | display-authors = 6 | title = Directing cellular information flow via CRISPR signal conductors | journal = Nature Methods | volume = 13| issue = 11| date = September 2016 | pmid = 27595406 | doi = 10.1038/nmeth.3994 | pages=938–944}}</ref>
CRISPR may revive the concept of [[Xenotransplantation|transplanting]] animal organs into people. [[Retroviruses]] present in animal genomes could harm transplant recipients. In 2015, a team eliminated 62 copies of a retrovirus's DNA from the pig genome in a kidney epithelial cell.<ref name="Science_Breakthrough"/> Researchers recently demonstrated the ability to birth live pig specimens after removing these retroviruses from their genome using CRISPR for the first time.<ref>{{Cite news|url=https://www.technologyreview.com/s/608579/crispr-opens-up-new-possibilities-for-transplants-using-pig-organs/|title=Using CRISPR on pigs could make their organs safer for human transplant|last=Mullin|first=Emily | name-list-format = vanc |work=MIT Technology Review|access-date=2017-09-09}}</ref>
CRISPR may have applications in tissue engineering and regenerative medicine, such as by creating human blood vessels that lack expression of [[MHC class II]] proteins, which often cause transplant rejection.<ref>{{cite journal | vauthors = Abrahimi P, Chang WG, Kluger MS, Qyang Y, Tellides G, Saltzman WM, Pober JS | title = Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9 | journal = Circulation Research | volume = 117 | issue = 2 | pages = 121–8 | date = July 2015 | pmid = 25940550 | pmc = 4490936 | doi = 10.1161/CIRCRESAHA.117.306290 }}</ref>
==== CRISPR in cancer ====
{{as of|2016}} CRISPR had been studied in animal models and cancer cell lines, to learn if it can be used to repair or thwart mutated genes that cause [[cancer]].<ref>{{cite journal | vauthors = Khan FA, Pandupuspitasari NS, Chun-Jie H, Ao Z, Jamal M, Zohaib A, Khan FA, Hakim MR, ShuJun Z | title = CRISPR/Cas9 therapeutics: a cure for cancer and other genetic diseases | journal = Oncotarget | volume = 7 | issue = 32 | pages = 52541–52552 | date = August 2016 | pmid = 27250031 | pmc = 5239572 | doi = 10.18632/oncotarget.9646 }}</ref>
The first clinical trial involving CRISPR started in 2016. It involved removing immune cells from people with lung cancer, using CRISPR to edit out the gene expressed PD-1, then administrating the altered cells back to the same person. 20 other trials were under way or nearly ready, mostly in China, {{as of|2017|lc=y}}.<ref>{{cite news | first = Michael | last = Le Page | name-list-format = vanc | title=Boom in human gene editing as 20 CRISPR trials gear up|url=https://www.newscientist.com/article/2133095-boom-in-human-gene-editing-as-20-crispr-trials-gear-up/|work=New Scientist|date=7 June 2017}}</ref>
In 2016, the [[Food and Drug Administration|United States Food and Drug Administration]] (FDA) approved a clinical trial in which CRISPR would be used to alter T cells extracted from people with different kinds of cancer and then administer those engineered T cells back to the same people.<ref>{{cite journal |doi=10.1038/nature.2016.20137 |title=First CRISPR clinical trial gets green light from US panel |journal=Nature |year=2016 |last1=Reardon |first1=Sara | name-list-format = vanc }}</ref>
In May 2018, the company CRISPR Therapeutics received approval to start a clinical trial with a CRISPR-based treatment for the blood disorder beta-thalassemia, which is scheduled to start in late 2018.<ref>{{Cite news|url=https://labiotech.eu/medical/crispr-therapeutics-clinical-trials/|title=CRISPR Therapeutics Plans First CRISPR Clinical Trial in Europe for 2018|date=2017-12-13|work=Labiotech.eu|access-date=2018-08-22 }}</ref><ref>{{Cite news|url=http://genomemag.com/2018/05/crispr-beta-thalassemia-treatment-approved-for-clinical-trial-in-europe/|title=CRISPR Beta-Thalassemia Treatment Approved for Clinical Trial in Europe|work=Genome Magazine|access-date=2018-08-22 }}</ref>
=== Gene function ===
In 2015, multiple studies attempted to systematically disable each individual human gene, in an attempt to identify which genes were essential to human biology. Between 1,600 and 1,800 genes passed this test—of the 20,000 or so known human genes. Such genes are more strongly activated, and unlikely to carry disabling mutations. They are more likely to have indispensable counterparts in other species. They build proteins that unite to form larger collaborative complexes. The studies also cataloged the essential genes in four cancer-cell lines and identified genes that are expendable in healthy cells, but crucial in specific tumor types and drugs that could target these rogue genes.<ref>{{cite web|title = The Revolutionary Gene-Editing Technique That Reveals Cancer's Weaknesses|url = https://www.theatlantic.com/science/archive/2015/11/a-revolutionary-gene-editing-technique-reveals-cancers-weaknesses/417495/|website = The Atlantic|access-date = 2016-02-21 | first = Ed | last = Yong | name-list-format = vanc | date = 2015-11-25}}</ref>
The specific functions of some 18 percent of the essential genes are unidentified. In one 2015 targeting experiment, disabling individual genes in groups of cells attempted to identify those involved in resistance to a [[melanoma]] drug. Each such gene manipulation is itself a separate "drug", potentially opening the entire genome to CRISPR-based regulation.<ref name="Science_Breakthrough" />
=== ''In vitro'' genetic depletion ===
Unenriched sequencing libraries often have abundant undesired sequences. Cas9 can specifically deplete the undesired sequences with double strand breakage with up to 99% efficiency and without significant [[Off-target genome editing|off-target effects]] as seen with [[restriction enzyme]]s. Treatment with Cas9 can deplete abundant rRNA while increasing pathogen sensitivity in RNA-seq libraries.<ref name="pmid26944702">{{cite journal | vauthors = Gu W, Crawford ED, O'Donovan BD, Wilson MR, Chow ED, Retallack H, DeRisi JL | title = Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications | journal = Genome Biology | volume = 17 | issue = | pages = 41 | date = March 2016 | pmid = 26944702 | pmc = 4778327 | doi = 10.1186/s13059-016-0904-5 }}</ref>
=== ''In vivo'' applications ===
CRISPR/Cas-9 can be used to edit the DNA of organisms ''in vivo'' and entire chromosomes can be eliminated from an organism at any point in its development. Chromosomes that have been deleted ''in vivo'' are the Y chromosomes and X chromosomes of adult lab mice and human chromosomes 14 and 21, in embryonic stem cell lines and [[aneuploid]] mice respectively. This method might be useful for treating genetic aneuploid diseases such as [[Down Syndrome]] and [[intersex]] disorders.<ref name="pmid29178945">{{cite journal | vauthors = Zuo E, Huo X, Yao X, Hu X, Sun Y, Yin J, He B, Wang X, Shi L, Ping J, Wei Y, Ying W, Wei W, Liu W, Tang C, Li Y, Hu J, Yang H | display-authors = 6 | title = CRISPR/Cas9-mediated targeted chromosome elimination | journal = Genome Biology | volume = 18 | issue = 1 | pages = 224 | year = 2017 | pmid = 29178945 | pmc = 5701507 | doi = 10.1186/s13059-017-1354-4 | lay-summary = https://www.genomeweb.com/gene-silencinggene-editing/crispr-used-eliminate-targeted-chromosomes-new-study | lay-source= Genome Web }}</ref>
Successful ''in vivo'' genome editing using CRISPR/Cas9 has been shown in several model organisms, such as ''Escherichia coli'',<ref name="Javed_2018">{{cite journal | vauthors = Javed MR, Sadaf M, Ahmed T, Jamil A, Nawaz M, Abbas H, Ijaz A | title = CRISPR-Cas System: History and Prospects as a Genome Editing Tool in Microorganisms | journal = Current Microbiology | volume = | issue = | pages = | date = August 2018 | pmid = 30078067 | doi = 10.1007/s00284-018-1547-4 | department = review }}</ref> ''Saccharomyces cerevisiae'',<ref name="Giersch_1017">{{cite journal | vauthors = Giersch RM, Finnigan GC | title = Yeast Still a Beast: Diverse Applications of CRISPR/Cas Editing Technology in S. cerevisiae | journal = The Yale Journal of Biology and Medicine | volume = 90 | issue = 4 | pages = 643–651 | date = December 2017 | pmid = 29259528 | pmc = 5733842 | doi = | url = }}</ref> ''Candida albicans'',<ref name="Raschmanová_2018">{{cite journal | vauthors = Raschmanová H, Weninger A, Glieder A, Kovar K, Vogl T | title = Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects | journal = Biotechnology Advances | volume = 36 | issue = 3 | pages = 641–665 | date = 2018 | pmid = 29331410 | doi = 10.1016/j.biotechadv.2018.01.006 | department = review }}</ref> ''Caenorhadbitis elegans'',<ref name="Ma_2015" /> ''Arabidopsis'',<ref name="Khurshid_2018">{{cite journal | vauthors = Khurshid H, Jan SA, Shinwari ZK, Jamal M, Shah SH | title = An Era of CRISPR/ Cas9 Mediated Plant Genome Editing | journal = Current Issues in Molecular Biology | volume = 26 | issue = | pages = 47–54 | date = 2018 | pmid = 28879855 | doi = 10.21775/cimb.026.047 | department = review }}</ref> ''Danio rerio'',<ref name="pmid30076892">{{cite journal | vauthors = Simone BW, Martínez-Gálvez G, WareJoncas Z, Ekker SC | title = Fishing for understanding: Unlocking the zebrafish gene editor's toolbox | journal = Methods | volume = | issue = | pages = | date = August 2018 | pmid = 30076892 | doi = 10.1016/j.ymeth.2018.07.012 | department = review }}</ref> ''Mus musculus''.<ref name="Singh_2015">{{cite journal | vauthors = Singh P, Schimenti JC, Bolcun-Filas E | title = A mouse geneticist's practical guide to CRISPR applications | journal = Genetics | volume = 199 | issue = 1 | pages = 1–15 | date = January 2015 | pmid = 25271304 | pmc = 4286675 | doi = 10.1534/genetics.114.169771 | department = review }}</ref><ref name="Soni_2018">{{cite journal | vauthors = Soni D, Wang DM, Regmi SC, Mittal M, Vogel SM, Schlüter D, Tiruppathi C | title = Deubiquitinase function of A20 maintains and repairs endothelial barrier after lung vascular injury | journal = Cell Death Discovery | volume = 4 | issue = 60 | pages = | date = May 2018 | pmid = | pmc = | doi = 10.1038/s41420-018-0056-3}}</ref> Successes have been achieved in the study of basic biology, in the creation of disease models,<ref name="Ma_2015">{{cite journal | vauthors = Ma D, Liu F | title = Genome Editing and Its Applications in Model Organisms | journal = Genomics, Proteomics & Bioinformatics | volume = 13 | issue = 6 | pages = 336–44 | date = December 2015 | pmid = 26762955 | pmc = 4747648 | doi = 10.1016/j.gpb.2015.12.001 | department = review }}</ref> and in the experimental treatment of disease models.<ref name="GaoTao2017">{{cite journal|display-authors=6|vauthors=Gao X, Tao Y, Lamas V, Huang M, Yeh WH, Pan B, Hu YJ, Hu JH, Thompson DB, Shu Y, Li Y, Wang H, Yang S, Xu Q, Polley DB, Liberman MC, Kong WJ, Holt JR, Chen ZY, Liu DR|date=December 2017|title=Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents|url=http://nrs.harvard.edu/urn-3:HUL.InstRepos:37298502|format=Submitted manuscript|journal=Nature|volume=553|issue=7687|pages=217–221|bibcode=2018Natur.553..217G|doi=10.1038/nature25164|pmc=5784267|pmid=29258297}}</ref>
Concerns have been raised that [[Off-target genome editing|off-target effects]] (editing of genes besides the ones intended) may obscure the results of a CRISPR gene editing experiment (the observed phenotypic change may not be due to modifying the target gene, but some other gene). Modifications to CRISPR have been made to minimize the possibility of off-target effects. In addition, orthogonal CRISPR experiments are recommended to confirm the results of a gene editing experiment.<ref name="Kadam_2018">{{cite journal | vauthors = Kadam US, Shelake RM, Chavhan RL, Suprasanna P | title = Concerns regarding 'off-target' activity of genome editing endonucleases | journal = Plant Physiology and Biochemistry | volume = 131 | issue = | pages = 22–30 | date = October 2018 | pmid = 29653762 | doi = 10.1016/j.plaphy.2018.03.027 | department = review }}</ref><ref name="Kimberland_2018">{{cite journal | vauthors = Kimberland ML, Hou W, Alfonso-Pecchio A, Wilson S, Rao Y, Zhang S, Lu Q | title = Strategies for controlling CRISPR/Cas9 off-target effects and biological variations in mammalian genome editing experiments | journal = Journal of Biotechnology | volume = 284 | issue = | pages = 91–101 | date = August 2018 | pmid = 30142414 | doi = 10.1016/j.jbiotec.2018.08.007 | department = review }}</ref>
== Patents and commercialization ==
{{as of|2014|December}}, [[patent rights]] to CRISPR were contested. Several companies formed to develop related drugs and research tools.<ref name="TR">{{cite web | url = http://www.technologyreview.com/featuredstory/532796/who-owns-the-biggest-biotech-discovery-of-the-century/ | title = Who Owns the Biggest Biotech Discovery of the Century? There's a bitter fight over the patents for CRISPR, a breakthrough new form of DNA editing | quote = CRISPR Patents Spark Fight to Control Genome Editing | website = MIT [[Technology Review]] | accessdate = 25 February 2015 }}</ref> As companies ramp up financing, doubts as to whether CRISPR can be quickly monetized were raised.<ref>{{cite web | last1 = Fye | first1 = Shaan | name-list-format = vanc | title=Genetic Rough Draft: Editas and CRISPR | url = http://atlasbusinessjournal.org/genetics/ | website = The Atlas Business Journal | accessdate = 19 January 2016 }}</ref> In February 2017 the US Patent Office ruled on a [[patent interference]] case brought by University of California with respect to patents issued to the [[Broad Institute]], and found that the Broad patents, with claims covering the application of CRISPR/cas9 in eukaryotic cells, were distinct from the inventions claimed by University of California.<ref>{{cite news | last1 = Pollack | first1 = Andrew | name-list-format = vanc | title = Harvard and M.I.T. Scientists Win Gene-Editing Patent Fight | url = https://www.nytimes.com/2017/02/15/science/broad-institute-harvard-mit-gene-editing-patent.html | work = The New York Times | date = 15 February 2017 }}</ref><ref>{{cite news | last1 = Akst | first1=Jef | name-list-format = vanc |title=Broad Wins CRISPR Patent Interference Case | url = http://www.the-scientist.com/?articles.view/articleNo/48490/title/Broad-Wins-CRISPR-Patent-Interference-Case | work = The Scientist Magazine | date=February 15, 2017 }}</ref><ref>{{cite news | last1 = Noonan | first1 = Kevin E.| name-list-format = vanc | title = PTAB Decides CRISPR Interference in Favor of Broad Institute -- Their Reasoning | url = http://www.patentdocs.org/2017/02/ptab-decides-crispr-interference-in-favor-of-broad-institute-their-reasoning.html | work = Patent Docs | date = February 16, 2017 }}</ref>
Shortly after, University of California filed an appeal of this ruling.<ref name="Potenza">{{cite news | last1 = Potenza | first1 = Alessandra | name-list-format = vanc | title = UC Berkeley challenges decision that CRISPR patents belong to Broad Institute 3 comments The legal fight will likely continue for months or even years | url = https://www.theverge.com/2017/4/13/15278478/crispr-gene-editing-tool-patent-dispute-appeal-ucb-mit-broad | accessdate=22 September 2017 | work=The Verge|date=April 13, 2017}}</ref><ref name="Buhr">{{cite news|last1=Buhr|first1=Sarah| name-list-format = vanc |title=The CRISPR patent battle is back on as UC Berkeley files an appeal|url=https://techcrunch.com/2017/07/26/the-crispr-patent-battle-is-back-on-as-uc-berkeley-files-an-appeal/|accessdate=22 September 2017|work=TechCrunch|date=July 26, 2017}}</ref>
{{as of|2013|November}}, SAGE Labs (part of [[Horizon Discovery]] group) had [[exclusive right]]s from one of those companies to produce and sell genetically engineered rats and non-exclusive rights for mouse and rabbit models.<ref>{{cite web | url = http://www.genengnews.com/insight-and-intelligence/crispr-madness/77899947/ | title = CRISPR Madness | website = GEN }}</ref> {{As of|2015|alt=By 2015}}, [[Thermo Fisher Scientific]] had licensed intellectual property from ToolGen to develop CRISPR reagent kits.<ref>{{Cite journal|last=Staff|date=1 April 2015|title=News: Products & Services|journal=[[Gen. Eng. Biotechnol. News|Genetic Engineering & Biotechnology News]]|type=Paper|volume=35|issue=7|page=8}}</ref>
In March 2017, the European Patent Office (EPO) announced its intention to allow broad claims for editing all kinds of cells to Max-Planck Institute in Berlin, University of California, and University of Vienna,<ref name="Philippidis"/><ref name="Akst">{{cite news|last1=Akst|first1=Jef| name-list-format = vanc |title=UC Berkeley Receives CRISPR Patent in Europe|url=http://www.the-scientist.com/?articles.view/articleNo/48987/title/UC-Berkeley-Receives-CRISPR-Patent-in-Europe/|accessdate=22 September 2017|work=The Scientist|date=March 24, 2017}}</ref> and in August 2017, the EPO announced its intention to allow CRISPR claims in a patent application that MilliporeSigma had filed.<ref name="Philippidis">{{cite news|last1=Philippidis|first1=Alex | name-list-format = vanc |title=MilliporeSigma to Be Granted European Patent for CRISPR Technology|work=Genetic Engineering & Biotechology News|date=August 7, 2017|url=http://www.genengnews.com/gen-news-highlights/milliporesigma-to-be-granted-european-patent-for-crispr-technology/81254776|accessdate=22 September 2017}}</ref> {{as of|2017|August}} the patent situation in Europe was complex, with MilliporeSigma, ToolGen, Vilnius University, and Harvard contending for claims, along with University of California and Broad.<ref>{{cite journal | last1 = Cohen | first1 = Jon | name-list-format = vanc | title = CRISPR patent battle in Europe takes a 'wild' twist with surprising player|journal=Science|date=4 August 2017 | doi = 10.1126/science.aan7211 | url = http://www.sciencemag.org/news/2017/08/crispr-patent-battle-europe-takes-wild-twist-surprising-player }}</ref>
As of November 2018, all the CRISPR patent holders and institutes associated with them have set up companies to commercialize the patents by sub-licensing them for therapeutic, agriculture and many other application areas to biotech firms, pharmaceuticals, agri-businesses etc. Caribou Biosciences, ERS Genomics, Editas Medicine, Intellia Therapeutics, and CRISPR Therapeutics are the spin offs associated with CRISPR landscape. Not many large scale commercial assignees have actively participated in the early phases of the CRISPR–Cas patent landscape. The only large establishments making it to the top ten are Dow AgroSciences and DuPont Nutrition Science (now merged as DowDuPont), together holding 20 inventions in CRISPR-Cas9 applications in agriculture and animal biotechnology<ref>{{Cite news|url=https://www.prweb.com/releases/2018/11/prweb15924785.htm|title=CRISPR Cas9 Genome Editing Market Worth $5.3 Billion by 2025|work=PRWeb|access-date=2018-11-30}}</ref>.
== Society and culture ==
=== Human germline modification ===
As of March 2015, multiple groups had announced ongoing research to learn how they one day might apply CRISPR to human embryos, including labs in the US, China, and the UK, as well as US biotechnology company [[OvaScience]].<ref>{{cite journal | first = Antonio | last = Regalado | name-list-format = vanc | journal = MIT Technology Review | date = March 5, 2015 | url = http://www.technologyreview.com/featuredstory/535661/engineering-the-perfect-baby/ | title = Engineering the Perfect Baby }}</ref> Scientists, including a CRISPR co-discoverer, urged a worldwide moratorium on applying CRISPR to the human germline, especially for clinical use. They said "scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans" until the full implications "are discussed among scientific and governmental organizations".<ref name="SCI-20150319">{{cite journal | vauthors = Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M, Greely HT, Jinek M, Martin GS, Penhoet E, Puck J, Sternberg SH, Weissman JS, Yamamoto KR | title = Biotechnology. A prudent path forward for genomic engineering and germline gene modification | journal = Science | volume = 348 | issue = 6230 | pages = 36–8 | date = April 2015 | pmid = 25791083 | pmc = 4394183 | doi = 10.1126/science.aab1028 | bibcode = 2015Sci...348...36B }}</ref><ref name="NAT-20150312">{{cite journal | vauthors = Lanphier E, Urnov F, Haecker SE, Werner M, Smolenski J | title = Don't edit the human germ line | journal = Nature | volume = 519 | issue = 7544 | pages = 410–1 | date = March 2015 | pmid = 25810189 | doi = 10.1038/519410a | bibcode = 2015Natur.519..410L }}</ref> These scientists support further low-level research on CRISPR and do not see CRISPR as developed enough for any clinical use in making heritable changes to humans.<ref name="NYT-20150319">{{cite news | last = Wade | first = Nicholas | name-list-format = vanc | title = Scientists Seek Ban on Method of Editing the Human Genome | url = https://www.nytimes.com/2015/03/20/science/biologists-call-for-halt-to-gene-editing-technique-in-humans.html | date =19 March 2015 | work = [[The New York Times]] | accessdate = 20 March 2015 | quote = The biologists writing in Science support continuing laboratory research with the technique, and few if any scientists believe it is ready for clinical use.}}</ref>
In April 2015, Chinese scientists reported results of an attempt to alter the [[DNA]] of non-viable [[human embryos]] using CRISPR to correct a [[mutation]] that causes [[beta thalassemia]], a lethal heritable disorder.<ref name="LiangXu2015">{{cite journal | vauthors = Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J | title = CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes | journal = Protein & Cell | volume = 6 | issue = 5 | pages = 363–72 | date = May 2015 | pmid = 25894090 | pmc = 4417674 | doi = 10.1007/s13238-015-0153-5 }}</ref><ref name="NYT-20150423">{{cite news | last = Kolata | first = Gina | name-list-format = vanc | title=Chinese Scientists Edit Genes of Human Embryos, Raising Concerns |url=https://www.nytimes.com/2015/04/24/health/chinese-scientists-edit-genes-of-human-embryos-raising-concerns.html |date=23 April 2015 |work=[[The New York Times]] |accessdate=24 April 2015 }}</ref> The study had previously been rejected by both ''[[Nature (journal)|Nature]]'' and ''[[Science (journal)|Science]]'' in part because of ethical concerns.<ref name=NatureNews>{{cite journal |doi=10.1038/nature.2015.17378 |title=Chinese scientists genetically modify human embryos |journal=Nature |year=2015 |last1=Cyranoski |first1=David |last2=Reardon |first2=Sara | name-list-format = vanc }}</ref> The experiments resulted in successfully changing only some of the intended genes, and had [[Off-target effects of genome editing|off-target effects]] on other genes. The researchers stated that CRISPR is not ready for clinical application in [[reproductive medicine]].<ref name=NatureNews/> In April 2016, Chinese scientists were reported to have made a second unsuccessful attempt to alter the DNA of non-viable human embryos using CRISPR - this time to alter the [[CCR5]] gene to make the embryo HIV resistant.<ref>{{Cite web|url=https://www.technologyreview.com/s/601235/chinese-researchers-experiment-with-making-hiv-proof-embryos/|title=Chinese Researchers Experiment with Making HIV-Proof Embryos|last=Regalado|first=Antonio|name-list-format = vanc |date=2016-05-08|website=MIT Technology Review|access-date=2016-06-10}}</ref>
In December 2015, an International Summit on Human Gene Editing took place in Washington under the guidance of [[David Baltimore]]. Members of national scientific academies of America, Britain and China discussed the ethics of germline modification. They agreed to support basic and clinical research under certain legal and ethical guidelines. A specific distinction was made between [[somatic cells]], where the effects of edits are limited to a single individual, versus germline cells, where genome changes could be inherited by descendants. Heritable modifications could have unintended and far-reaching consequences for human evolution, genetically (e.g. gene/environment interactions) and culturally (e.g. [[Social Darwinism]]). Altering of [[gametocytes]] and embryos to generate inheritable changes in humans was defined to be irresponsible. The group agreed to initiate an international forum to address such concerns and harmonize regulations across countries.<ref name="National Academy of Science">{{cite web | url = http://www8.nationalacademies.org/onpinews/newsitem.aspx?RecordID=12032015a | date =3 December 2015 | title = International Summit on Gene Editing | publisher =[[National Academies of Sciences, Engineering, and Medicine]] | accessdate = 3 December 2015 }}</ref>
In November 2018, [[He Jiankui|Jiankui He]] announced that he had edited two human embryos, to attempt to disable the gene for [[CCR5]], which codes for a receptor that [[HIV]] uses to enter cells. He said that twin girls, Lulu and Nana, had been born a few weeks earlier. He said that the girls still carried functional copies of CCR5 along with disabled CCR5 ([[mosaicism]]) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature.<ref>{{cite news |last1=Begley |first1=Sharon | name-list-format = vanc |title=Amid uproar, Chinese scientist defends creating gene-edited babies |url=https://www.statnews.com/2018/11/28/chinese-scientist-defends-creating-gene-edited-babies/ |work=STAT |date=28 November 2018}}</ref>
=== Policy barriers to genetic engineering ===
Policy regulations for the CRISPR/cas9 system vary around the globe. In February 2016, British scientists were given permission by regulators to genetically modify [[human embryos]] by using CRISPR-Cas9 and related techniques. However, researchers were forbidden from implanting the embryos and the embryos were to be destroyed after seven days.<ref>{{cite journal | vauthors = Callaway E | title = UK scientists gain licence to edit genes in human embryos | journal = Nature | volume = 530 | issue = 7588 | pages = 18 | date = February 2016 | pmid = 26842037 | doi = 10.1038/nature.2016.19270 | bibcode = 2016Natur.530...18C }}</ref>
The US has an elaborate, interdepartmental regulatory system to evaluate new genetically modified foods and crops. For example, the [[Agriculture Risk Protection Act of 2000]] gives the [[United States Department of Agriculture|USDA]] the authority to oversee the detection, control, eradication, suppression, prevention, or retardation of the spread of plant pests or noxious weeds to protect the agriculture, environment and economy of the US. The act regulates any [[genetically modified organism]] that utilizes the genome of a predefined "plant pest" or any plant not previously categorized.<ref>{{cite journal | vauthors = McHughen A, Smyth S | title = US regulatory system for genetically modified [genetically modified organism (GMO), rDNA or transgenic] crop cultivars | journal = Plant Biotechnology Journal | volume = 6 | issue = 1 | pages = 2–12 | date = January 2008 | pmid = 17956539 | doi = 10.1111/j.1467-7652.2007.00300.x }}</ref> In 2015, Yinong Yang successfully deactivated 16 specific genes in the white button mushroom, to make them non-browning. Since he had not added any foreign-species ([[transgenic]]) DNA to his organism, the mushroom could not be regulated by the USDA under Section 340.2.<ref>{{cite web|url=https://www.aphis.usda.gov/biotechnology/downloads/reg_loi/15-321-01_air_response_signed.pdf|title=Re: Request to confirm |author=USDA| authorlink=USDA}}</ref> Yang's white button mushroom was the first organism genetically modified with the Crispr/cas9 protein system to pass US regulation.<ref name="Waltz_2016">{{cite journal | vauthors = Waltz E | title = Gene-edited CRISPR mushroom escapes US regulation | journal = Nature | volume = 532 | issue = 7599 | pages = 293 | year = 2016 | pmid = 27111611 | doi = 10.1038/nature.2016.19754 | bibcode = 2016Natur.532..293W }}</ref> In 2016, the USDA sponsored a committee to consider future regulatory policy for upcoming genetic modification techniques. With the help of the US [[National Academies of Sciences, Engineering, and Medicine|National Academies of Sciences, Engineering and Medicine]], special interests groups met on April 15 to contemplate the possible advancements in genetic engineering within the next five years and any new regulations that might be needed as a result.<ref>{{cite journal | vauthors = Ledford H | title = Gene-editing surges as US rethinks regulations | journal = Nature | volume = 532 | issue = 7598 | pages = 158–9 | date = April 2016 | pmid = 27075074 | doi = 10.1038/532158a | bibcode = 2016Natur.532..158L }}</ref> The [[FDA]] in 2017 proposed a rule that would classify genetic engineering modifications to animals as "animal drugs", subjecting them to strict regulation if offered for sale, and reducing the ability for individuals and small businesses to make them profitably.<ref>[http://www.gizmodo.com.au/2017/02/the-fda-is-cracking-down-on-rogue-genetic-engineers/ "The FDA Is Cracking Down On Rogue Genetic Engineers"], Kristen V. Brown. Gizmodo. February 1, 2017. Retrieved 5 feb 2017</ref><ref>{{cite web|url=https://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/UCM113903.pdf|title=Guidance for Industry #187 / Regulation of Intentionally Altered Genomic DNA in Animals |publisher=}}</ref>
In China, where social conditions sharply contrast with the west, genetic diseases carry a heavy stigma.<ref name="Cyranoski_2017">{{cite journal | vauthors = Cyranoski D | title = China's embrace of embryo selection raises thorny questions | journal = Nature | volume = 548 | issue = 7667 | pages = 272–274 | year = 2017 | pmid = 28816265 | doi = 10.1038/548272a | bibcode = 2017Natur.548..272C }}</ref> This leaves China with fewer policy barriers to the use of this technology.<ref name="Peng_2016">{{cite journal | vauthors = Peng Y | title = The morality and ethics governing CRISPR-Cas9 patents in China | journal = Nature Biotechnology | volume = 34 | issue = 6 | pages = 616–8 | year = 2016 | pmid = 27281418 | doi = 10.1038/nbt.3590 }}</ref><ref name = "WSJ_2018">{{cite news | url = https://www.wsj.com/articles/china-unhampered-by-rules-races-ahead-in-gene-editing-trials-1516562360 | title = China, Unhampered by Rules, Races Ahead in Gene-Editing Trials | last1 = Rana | first1 = Preetika | last2 = Marcus | first2 = Amy Dockser | last3 = Fan | first3 = Wenxin | name-list-format = vanc | date = 2018-01-21 | work = Wall Street Journal | access-date = 2018-01-23 | issn = 0099-9660 }}</ref>
=== Recognition ===
In 2012, and 2013, CRISPR was a runner-up in ''[[Science Magazine]]''<nowiki/>'s [[Breakthrough of the Year]] award. In 2015, it was the winner of that award.<ref name="Science_Breakthrough"/> CRISPR was named as one of ''[[MIT Technology Review]]''{{'}}s 10 breakthrough technologies in 2014 and 2016.<ref name="MITCRISPR">{{cite news | last1 = Talbot | first1 = David | name-list-format = vanc | title = Precise Gene Editing in Plants/ 10 Breakthrough Technologies 2016 |url=https://www.technologyreview.com/s/600765/10-breakthrough-technologies-2016-precise-gene-editing-in-plants/ |accessdate=18 March 2016 | work = MIT Technology review | publisher = Massachusetts Institute of Technology|date=2016}}</ref><ref name="MITGene">{{cite news | last1 = Larson | first1 = Christina | last2 = Schaffer | first2 = Amanda | name-list-format = vanc | title=Genome Editing/ 10 Breakthrough Technologies 2014 | url =https://www.technologyreview.com/s/526511/genome-editing/ | accessdate=18 March 2016 | publisher =Massachusetts Institute of Technology | date = 2014 }}</ref> In 2016, [[Jennifer Doudna]], [[Emmanuelle Charpentier]], along with Rudolph Barrangou, [[Philippe Horvath]], and [[Feng Zhang]] won the Gairdner International award. In 2017, Jennifer Doudna and Emmanuelle Charpentier were awarded the Japan Prize for their revolutionary invention of CRISPR-Cas9 in Tokyo, Japan. In 2016, Emmanuelle Charpentier, Jennifer Doudna, and Feng Zhang won the [[Tang Prize]] in Biopharmaceutical Science.<ref>{{Cite web|url=http://www.tang-prize.org/en/owner_detail.php?cat=11&id=554|title=Tang Prize Laureates|vauthors =((良艮創意,很好設計,李維宗設計))|website=www.tang-prize.org|language=en|access-date=2018-08-05}}</ref>
== See also ==
{{div col|colwidth=18em}}
* [[CRISPR gene editing]]
* [[Genetics]]
* [[Glossary of genetics]]
|