View source for CRISPR

{{short description|Family of DNA sequences found in prokaryotic organisms}}
{{cs1 config|name-list-style=vanc|display-authors=6}}
{{About|the prokaryotic antiviral system|the use in editing genes|CRISPR gene editing}}
{{Infobox nonhuman protein
| Name = Cascade (CRISPR-associated complex for antiviral defense)
| image = 4QYZ.png
| caption = CRISPR Cascade protein (cyan) bound to CRISPR RNA (green) and phage DNA (red)<ref name="Mulepati_2014">{{PDB|4QYZ}}: {{cite journal | vauthors = Mulepati S, Héroux A, Bailey S| title = Crystal structure of a CRISPR RNA–guided surveillance complex bound to a ssDNA target| journal = Science | volume = 345 | issue = 6203 | pages = 1479–1484 | year = 2014 | pmid = 25123481 | pmc = 4427192| doi = 10.1126/science.1256996 | bibcode = 2014Sci...345.1479M}}</ref>
| Organism = ''Escherichia coli''
| TaxID = 511145
| Symbol = CRISPR
| EntrezGene = 947229
| HomoloGene = 
| PDB = 4QYZ
| UniProt = P38036
| RefSeqmRNA = 
| RefSeqProtein = NP_417241.1
}}
[[File:Crispr.png|thumb|262px|Diagram of the CRISPR prokaryotic antiviral defense mechanism<ref name="Horvath_2010">{{cite journal | vauthors = Horvath P, Barrangou R | author-link2=Rodolphe Barrangou | title = CRISPR/Cas, the immune system of bacteria and archaea | journal = Science | volume = 327 | issue = 5962 | pages = 167–170 | date = January 2010 | pmid = 20056882 | doi = 10.1126/science.1179555 | bibcode=2010Sci...327..167H }}</ref>]]
'''CRISPR''' ({{IPAc-en|ˈ|k|ɹ|ɪ|s|p|ɚ}}; acronym of '''clustered regularly interspaced short palindromic repeats''') is a family of [[DNA]] sequences found in the [[genome]]s of [[prokaryotic]] organisms such as [[bacteria]] and [[archaea]].<ref name="pmid25574773"/> Each sequence within an individual prokaryotic CRISPR is derived from a DNA fragment of a [[bacteriophage]] that had previously infected the prokaryote or one of its ancestors.<ref name="Hille2018"/><ref name="Rath_2015">{{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 | pages = 119–128 | date = October 2015 | pmid = 25868999 | doi = 10.1016/j.biochi.2015.03.025 | doi-access = free }}</ref> These sequences are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral (i.e. anti-[[Bacteriophage|phage]]) defense system of prokaryotes and provide a form of heritable,<ref name="Hille2018"/> [[acquired immunity]].<ref name="pmid25574773">{{cite journal | vauthors = Barrangou R | author-link=Rodolphe Barrangou | title = The roles of CRISPR-Cas systems in adaptive immunity and beyond | journal = Current Opinion in Immunology | volume = 32 | pages = 36–41 | year = 2015 | pmid = 25574773 | doi = 10.1016/j.coi.2014.12.008 }}</ref><ref name="Redman_2016">{{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 = 213–215 | 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 | author-link1=Rodolphe Barrangou | title = CRISPR provides acquired resistance against viruses in prokaryotes | journal = Science | volume = 315 | issue = 5819 | pages = 1709–1712 | date = March 2007 | pmid = 17379808 | doi = 10.1126/science.1138140 | bibcode = 2007Sci...315.1709B | hdl=20.500.11794/38902 | hdl-access = free }} {{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 = 1843–1845 | date = December 2008 | pmid = 19095942 | pmc = 2695655 | doi = 10.1126/science.1165771 | bibcode = 2008Sci...322.1843M }}</ref> CRISPR is 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 |hdl=21.11116/0000-0003-FC0D-4 |hdl-access=free }}</ref>

[[Cas9]] (or "CRISPR-associated protein 9") is an [[enzyme]] that uses CRISPR sequences as a guide to recognize and open up specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as [[CRISPR gene editing|CRISPR-Cas9]] that can be used to edit genes within living organisms.<ref>{{cite journal | vauthors = Bak RO, Gomez-Ospina N, Porteus MH | title = Gene Editing on Center Stage | journal = Trends in Genetics | volume = 34 | issue = 8 | pages = 600–611 | date = August 2018 | pmid = 29908711 | doi = 10.1016/j.tig.2018.05.004 }}</ref><ref name="pmid24651067">{{cite journal | vauthors = Zhang F, Wen Y, Guo X | title = CRISPR/Cas9 for genome editing: progress, implications and challenges | journal = Human Molecular Genetics | volume = 23 | issue = R1 | pages = R40–6 | year = 2014 | pmid = 24651067 | doi = 10.1093/hmg/ddu125 | doi-access = free }}</ref> This editing process has a wide variety of applications including basic [[Biology|biological]] research, development of [[biotechnology|biotechnological]] products, and treatment of diseases.<ref name="Proteintech">CRISPR-CAS9, TALENS and ZFNS – the battle in gene editing https://www.ptglab.com/news/blog/crispr-cas9-talens-and-zfns-the-battle-in-gene-editing/ {{Webarchive|url=https://web.archive.org/web/20210525031855/https://www.ptglab.com/news/blog/crispr-cas9-talens-and-zfns-the-battle-in-gene-editing/ |date=2021-05-25 }}</ref><ref name="Hsu2014"/> The development of the CRISPR-Cas9 genome editing technique was recognized by the [[Nobel Prize in Chemistry]] in 2020 awarded to [[Emmanuelle Charpentier]] and [[Jennifer Doudna]].<ref name="Nobel_Foundation">{{cite web |title=Press release: The Nobel Prize in Chemistry 2020 |url=https://www.nobelprize.org/prizes/chemistry/2020/press-release/ |publisher=Nobel Foundation |access-date=7 October 2020 |archive-date=15 January 2021 |archive-url=https://web.archive.org/web/20210115172618/https://www.nobelprize.org/prizes/chemistry/2020/press-release/ |url-status=live }}</ref><ref name="NYT-20201007">{{cite news |vauthors=Wu KJ, Peltier E |title=Nobel Prize in Chemistry Awarded to 2 Scientists for Work on Genome Editing – Emmanuelle Charpentier and Jennifer A. Doudna developed the Crispr tool, which can alter the DNA of animals, plants and microorganisms with high precision. |url=https://www.nytimes.com/2020/10/07/science/nobel-prize-chemistry-crispr.html |date=7 October 2020 |work=[[The New York Times]] |access-date=7 October 2020 |archive-date=8 October 2020 |archive-url=https://web.archive.org/web/20201008094313/https://www.nytimes.com/2020/10/07/science/nobel-prize-chemistry-crispr.html |url-status=live }}</ref>

== History ==
=== Repeated sequences ===
The discovery of clustered DNA repeats took place independently in three parts of the world. The first description of what would later be called CRISPR is from [[Osaka University]] researcher [[Yoshizumi Ishino]] and his colleagues in 1987. They accidentally cloned part of a CRISPR sequence together with the "''iap" gene'' ''(isozyme conversion of alkaline phosphatase)'' from their target genome, that of ''[[Escherichia coli]]''.<ref name="Rawat_2021">{{cite journal | vauthors = Rawat A, Roy M, Jyoti A, Kaushik S, Verma K, Srivastava VK | title = Cysteine proteases: Battling pathogenic parasitic protozoans with omnipresent enzymes | journal = Microbiological Research | volume = 249 | page = 126784 | date = August 2021 | pmid = 33989978 | doi = 10.1016/j.micres.2021.126784 | doi-access = free }}</ref><ref name="Ishino-1987">{{cite journal | vauthors = Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A | title = Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product | journal = Journal of Bacteriology | volume = 169 | issue = 12 | pages = 5429–5433 | date = December 1987 | pmid = 3316184 | pmc = 213968 | doi = 10.1128/jb.169.12.5429-5433.1987 }}</ref> The organization of the repeats was unusual. Repeated sequences are typically arranged consecutively, without interspersing different sequences.<ref name="Hsu2014"/><ref name="Ishino-1987"/> They did not know the function of the interrupted clustered repeats.

In 1993, researchers of ''[[Mycobacterium tuberculosis]]'' in the Netherlands published two articles about a cluster of interrupted [[direct repeat]]s (DR) in that bacterium. They recognized the diversity of the sequences that intervened in the direct repeats among different strains of ''M. tuberculosis''<ref name="Soolingen1993">{{cite journal | vauthors = van Soolingen D, de Haas PE, Hermans PW, Groenen PM, van Embden JD | title = Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis | journal = Journal of Clinical Microbiology | volume = 31 | issue = 8 | pages = 1987–1995 | date = August 1993 | pmid = 7690367 | pmc = 265684 | doi = 10.1128/JCM.31.8.1987-1995.1993 }}</ref> and used this property to design a typing method called ''[[wikt:spoligotyping|spoligotyping]]'', still in use today.<ref name="Groenen1993">{{cite journal | vauthors = Groenen PM, Bunschoten AE, van Soolingen D, van Embden JD | title = Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method | journal = Molecular Microbiology | volume = 10 | issue = 5 | pages = 1057–1065 | date = December 1993 | pmid = 7934856 | doi = 10.1111/j.1365-2958.1993.tb00976.x }}</ref><ref name="Mojica2016">{{cite journal |vauthors=Mojica FJ, Montoliu L |title=On the Origin of CRISPR-Cas Technology: From Prokaryotes to Mammals |journal=Trends in Microbiology |volume=24 |issue=10 |pages=811–820 |year=2016 |pmid=27401123 |doi=10.1016/j.tim.2016.06.005 }}</ref>

[[Francisco Mojica]] at the [[University of Alicante]] in Spain studied the function of repeats in the archaeal species ''[[Haloferax]]'' and ''[[Haloarcula]]''. Mojica's supervisor surmised that the clustered repeats had a role in correctly segregating replicated DNA into daughter cells during cell division, because plasmids and chromosomes with identical repeat arrays could not coexist in ''[[Haloferax volcanii]]''. Transcription of the interrupted repeats was also noted for the first time; this was the first full characterization of CRISPR.<ref name="Mojica2016"/><ref name="Mojica2016b">{{cite journal |vauthors=Mojica FJ, Rodriguez-Valera F |title=The discovery of CRISPR in archaea and bacteria |journal=The FEBS Journal |volume=283 |issue=17 |pages=3162–3169 |year=2016 |pmid=27234458 |doi=10.1111/febs.13766 |hdl=10045/57676 |hdl-access=free }}</ref> By 2000, Mojica and his students, after an automated search of published genomes, identified interrupted repeats in 20 species of microbes as belonging to the same family.<ref name="Mojica_2000">{{cite journal | vauthors = Mojica FJ, Díez-Villaseñor C, Soria E, Juez G | title = Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria | journal = Molecular Microbiology | volume = 36 | issue = 1 | pages = 244–246 | date = April 2000 | pmid = 10760181 | doi = 10.1046/j.1365-2958.2000.01838.x |doi-access=free}}</ref> Because those sequences were interspaced, Mojica initially called these sequences "short regularly spaced repeats" (SRSR).<ref name="Isaacson_2021">{{cite book |vauthors=Isaacson W |author-link1=Walter Isaacson |title=The Code Breaker: Jennifer Doudna, Gene Editing, and the Future of the Human Race |url=https://books.google.com/books?id=f_D3DwAAQBAJ |page=73 |location=New York |publisher=Simon & Schuster |year=2021 |isbn=978-1-9821-1585-2 |oclc=1239982737 |access-date=2021-10-20 |archive-date=2023-01-14 |archive-url=https://web.archive.org/web/20230114155931/https://books.google.com/books?id=f_D3DwAAQBAJ |url-status=live }}</ref> In 2001, Mojica and [[Ruud Jansen]], who were searching for additional interrupted repeats, proposed the acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to unify the numerous acronyms used to describe these sequences.<ref name="Mojica2016b"/><ref name="Barrangou_2013">{{cite book | vauthors = Barrangou R, van der Oost J | author-link1 = Rodolphe Barrangou | title = CRISPR-Cas Systems: RNA-mediated Adaptive Immunity in Bacteria and Archaea | date = 2013 | publisher = Springer | location = Heidelberg | isbn = 978-3-642-34656-9 | page = 6 }}</ref> In 2002, Tang, et al. showed evidence that CRISPR repeat regions from the genome of ''[[Archaeoglobus fulgidus]]'' were transcribed into long RNA molecules subsequently processed into unit-length small RNAs, plus some longer forms of 2, 3, or more spacer-repeat units.<ref name="pmid12032318">{{cite journal | vauthors = Tang TH, Bachellerie JP, Rozhdestvensky T, Bortolin ML, Huber H, Drungowski M, Elge T, Brosius J, Hüttenhofer A | title = Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 99 | issue = 11 | pages = 7536–7541 | date = May 2002 | pmid = 12032318 | pmc = 124276 | doi = 10.1073/pnas.112047299 | doi-access = free | bibcode = 2002PNAS...99.7536T }}</ref><ref name="pmid25994611">{{cite journal | vauthors = Charpentier E, Richter H, van der Oost J, White MF | title = Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity | journal = FEMS Microbiology Reviews | volume = 39 | issue = 3 | pages = 428–441 | date = May 2015 | pmid = 25994611 | pmc = 5965381 | doi = 10.1093/femsre/fuv023 }}</ref>

In 2005, [[yogurt]] researcher [[Rodolphe Barrangou]] discovered that ''[[Streptococcus thermophilus]]'', after iterative phage infection challenges, develops increased phage resistance due to the incorporation of additional CRISPR spacer sequences.<ref name="Romero_2020">{{cite journal | vauthors = Romero DA, Magill D, Millen A, Horvath P, Fremaux C | title = Dairy lactococcal and streptococcal phage-host interactions: an industrial perspective in an evolving phage landscape | journal = FEMS Microbiology Reviews | volume = 44 | issue = 6 | pages = 909–932 | date = November 2020 | pmid = 33016324 | doi = 10.1093/femsre/fuaa048 | doi-access =  }}</ref> Barrangou's employer, the Danish food company Danisco, then developed phage-resistant ''S. thermophilus'' strains for yogurt production. Danisco was later bought by [[DuPont]], which owns about 50 percent of the global dairy culture market, and the technology spread widely.<ref name="rbpont">{{cite news |vauthors=Molteni M, Huckins G |title=The WIRED Guide to Crispr |url=https://www.wired.com/story/wired-guide-to-crispr/ |agency=Wired Magazine |publisher=Condé Nast |date=1 August 2020 |access-date=23 February 2021 |archive-date=23 October 2021 |archive-url=https://web.archive.org/web/20211023062846/https://www.wired.com/story/wired-guide-to-crispr/ |url-status=live }}</ref>

=== CRISPR-associated systems ===
A major advance in understanding CRISPR came with Jansen's observation that the prokaryote repeat cluster was accompanied by four homologous genes that make up CRISPR-associated systems, ''cas'' 1–4. The Cas proteins showed [[helicase]] and [[nuclease]] [[Structural motif|motifs]], suggesting a role in the dynamic structure of the CRISPR loci.<ref name="pmid11952905">{{cite journal | vauthors = Jansen R, Embden JD, Gaastra W, Schouls LM | title = Identification of genes that are associated with DNA repeats in prokaryotes | journal = Molecular Microbiology | volume = 43 | issue = 6 | pages = 1565–1575 | date = March 2002 | pmid = 11952905 | doi = 10.1046/j.1365-2958.2002.02839.x | doi-access = free }}</ref> In this publication, the acronym CRISPR was used as the universal name of this pattern, but its function remained enigmatic.

[[File:SimpleCRISPR.jpg|thumb|Simplified diagram of a CRISPR locus. The three major components of a CRISPR locus are shown: ''cas'' genes, a leader sequence, and a repeat-spacer array. Repeats are shown as gray boxes and spacers are colored bars. The arrangement of the three components is not always as shown.<ref name="pmid20056882">{{cite journal | vauthors = Horvath P, Barrangou R | author-link2=Rodolphe Barrangou | title = CRISPR/Cas, the immune system of bacteria and archaea | journal = Science | volume = 327 | issue = 5962 | pages = 167–170 | date = January 2010 | pmid = 20056882 | doi = 10.1126/Science.1179555 | bibcode = 2010Sci...327..167H }}</ref><ref name="pmid20125085">{{cite journal | vauthors = Marraffini LA, Sontheimer EJ | title = CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea | journal = Nature Reviews Genetics | volume = 11 | issue = 3 | pages = 181–190 | date = March 2010 | pmid = 20125085 | pmc = 2928866 | doi = 10.1038/nrg2749 }}</ref>
In addition, several CRISPRs with similar sequences can be present in a single genome, only one of which is associated with ''cas'' genes.<ref name="pmid17521438">{{cite journal | vauthors = Grissa I, Vergnaud G, Pourcel C | title = The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats | journal = BMC Bioinformatics | volume = 8 | page = 172 | date = May 2007 | pmid = 17521438 | pmc = 1892036 | doi = 10.1186/1471-2105-8-172 | doi-access = free }}</ref>]]
In 2005, three independent research groups showed that some CRISPR spacers are derived from [[phage]] DNA and [[extrachromosomal DNA]] such as [[plasmid]]s.<ref name="pmid15758212">{{cite journal | vauthors = Pourcel C, Salvignol G, Vergnaud G | title = CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies | journal = Microbiology | volume = 151 | issue = Pt 3 | pages = 653–663 | date = March 2005 | pmid = 15758212 | doi = 10.1099/mic.0.27437-0 | doi-access = free }}</ref><ref name="pmid15791728">{{cite journal | vauthors = Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E | title = Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements | journal = Journal of Molecular Evolution | volume = 60 | issue = 2 | pages = 174–182 | date = February 2005 | pmid = 15791728 | doi = 10.1007/s00239-004-0046-3 | bibcode = 2005JMolE..60..174M }}</ref><ref name="pmid16079334">{{cite journal | vauthors = Bolotin A, Quinquis B, Sorokin A, Ehrlich SD | title = Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin | journal = Microbiology | volume = 151 | issue = Pt 8 | pages = 2551–2561 | date = August 2005 | pmid = 16079334 | doi = 10.1099/mic.0.28048-0 |doi-access=free}}</ref> In effect, the spacers are fragments of DNA gathered from viruses that previously attacked the cell. The source of the spacers was a sign that the CRISPR-''cas'' system could have a role in adaptive immunity in [[bacteria]].<ref name="pmid20056882"/><ref name="pmid25963251">{{cite journal | vauthors = Morange M | title = What history tells us XXXVII. CRISPR-Cas: The discovery of an immune system in prokaryotes | journal = Journal of Biosciences | volume = 40 | issue = 2 | pages = 221–223 | date = June 2015 | pmid = 25963251 | doi = 10.1007/s12038-015-9532-6 | doi-access = free }}</ref> All three studies proposing this idea were initially rejected by high-profile journals, but eventually appeared in other journals.<ref name="Lander_2016">{{cite journal | vauthors = Lander ES | title = The Heroes of CRISPR | journal = Cell | volume = 164 | issue = 1–2 | pages = 18–28 | date = January 2016 | pmid = 26771483 | doi = 10.1016/j.cell.2015.12.041 | doi-access = free }}</ref>

The first publication<ref name="pmid15791728"/> proposing a role of CRISPR-Cas in microbial immunity, by Mojica and collaborators at the [[University of Alicante]], predicted a role for the RNA transcript of spacers on target recognition in a mechanism that could be analogous to the [[RNA interference]] system used by eukaryotic cells. Koonin and colleagues extended this RNA interference hypothesis by proposing mechanisms of action for the different CRISPR-Cas subtypes according to the predicted function of their proteins.<ref name="Makarova2006">{{cite journal | vauthors = Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV | author-link5=Eugene Koonin | title = A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action | journal = Biology Direct | volume = 1 | page = 7 | date = March 2006 | pmid = 16545108 | pmc = 1462988 | doi = 10.1186/1745-6150-1-7 | doi-access=free }}</ref>

Experimental work by several groups revealed the basic mechanisms of CRISPR-Cas immunity. In 2007, the first experimental evidence that CRISPR was an adaptive immune system was published.<ref name="pmid17379808"/><ref name="Hsu2014"/> A CRISPR region in ''[[Streptococcus thermophilus]]'' acquired spacers from the DNA of an infecting [[bacteriophage]]. The researchers manipulated the resistance of ''S. thermophilus'' to different types of phages by adding and deleting spacers whose sequence matched those found in the tested phages.<ref name="craze">{{cite journal | vauthors = Pennisi E | author-link = Elizabeth Pennisi | title = The CRISPR craze | journal = Science | volume = 341 | issue = 6148 | pages = 833–836 | date = August 2013 | pmid = 23970676 | doi = 10.1126/science.341.6148.833 | department = News Focus | bibcode = 2013Sci...341..833P }}</ref><ref name="Marraffini2015"/> In 2008, Brouns and Van der Oost identified a complex of Cas proteins called Cascade, that in ''E. coli'' cut the CRISPR RNA precursor within the repeats into mature spacer-containing RNA molecules called [[CRISPR RNA]] (crRNA), which remained bound to the protein complex.<ref name="Brouns_2008">{{cite journal | vauthors = Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J | author-link9=Eugene Koonin | title = Small CRISPR RNAs guide antiviral defense in prokaryotes | journal = Science | volume = 321 | issue = 5891 | pages = 960–964 | date = August 2008 | pmid = 18703739 | pmc = 5898235 | doi = 10.1126/science.1159689 | bibcode = 2008Sci...321..960B }}</ref> Moreover, it was found that Cascade, crRNA and a helicase/nuclease ([[Cas3]]) were required to provide a bacterial host with immunity against infection by a [[DNA virus]]. By designing an anti-virus CRISPR, they demonstrated that two orientations of the crRNA (sense/antisense) provided immunity, indicating that the crRNA guides were targeting [[dsDNA]]. That year Marraffini and Sontheimer confirmed that a CRISPR sequence of ''[[S. epidermidis]]'' targeted DNA and not RNA to prevent [[Bacterial conjugation|conjugation]]. This finding was at odds with the proposed RNA-interference-like mechanism of CRISPR-Cas immunity, although a CRISPR-Cas system that targets foreign RNA was later found in ''[[Pyrococcus furiosus]]''.<ref name="Hsu2014"/><ref name="Marraffini2015"/> A 2010 study showed that CRISPR-Cas cuts strands of both phage and plasmid DNA in ''S. thermophilus''.<ref name="Garneau2010">{{cite journal | vauthors = Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S | author-link5=Rodolphe Barrangou | title = The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA | journal = Nature | volume = 468 | issue = 7320 | pages = 67–71 | date = November 2010 | pmid = 21048762 | doi = 10.1038/nature09523 | bibcode = 2010Natur.468...67G }}</ref>

=== Cas9 ===
{{main|Cas9}}
[[File:Cas12a vs Cas9 cleavage position.svg|thumb|A diagram of the CRISPR nucleases [[Cas12a]] and [[Cas9]] with the position of DNA cleavage shown relative to their [[Protospacer adjacent motif|PAM sequences]] in a zoom-in]] 
A simpler CRISPR system from ''[[Streptococcus pyogenes]]'' uses the protein [[Cas9]], an endonuclease functioning with two small RNAs—crRNA and tracrRNA—to form a four-component complex.<ref name="Deltcheva2011">{{cite journal | vauthors = Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E | title = CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III | journal = Nature | volume = 471 | issue = 7340 | pages = 602–607 | date = March 2011 | pmid = 21455174 | pmc = 3070239 | doi = 10.1038/nature09886 | bibcode = 2011Natur.471..602D }}</ref><ref name="Barrangou2015">{{cite journal | vauthors = Barrangou R | author-link=Rodolphe Barrangou | title = Diversity of CRISPR-Cas immune systems and molecular machines | journal = Genome Biology | volume = 16 | page = 247 | date = November 2015 | pmid = 26549499 | pmc = 4638107 | doi = 10.1186/s13059-015-0816-9 | doi-access=free }}</ref> In 2012, [[Jennifer Doudna]] and [[Emmanuelle Charpentier]] simplified this into a two-component system by fusing the RNAs into a "[[Guide_RNA#Structure|single-guide RNA]]", enabling Cas9 to target and cut specific DNA sequences—a breakthrough that earned them the [[Nobel Prize in Chemistry]] in 2020.<ref name="pmid22745249">{{cite journal | vauthors = Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E | author-link5=Jennifer Doudna | title = A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity | journal = Science | volume = 337 | issue = 6096 | pages = 816–821 | date = August 2012 | pmid = 22745249 | pmc =  6286148| doi = 10.1126/science.1225829 | bibcode = 2012Sci...337..816J }}</ref> Parallel work showed the ''S. thermophilus'' Cas9 could be similarly reprogrammed by altering the crRNA sequence.<ref name="Mojica2016"/> These developments spurred genome editing efforts, including demonstrations by groups led by [[Feng Zhang]] and [[George M. Church|George Church]] showing genome editing in human cells using CRISPR-Cas9.<ref name="Hsu2014">{{cite journal | vauthors = Hsu PD, Lander ES, Zhang F | author-link3=Feng Zhang | title = Development and applications of CRISPR-Cas9 for genome engineering | journal = Cell | volume = 157 | issue = 6 | pages = 1262–1278 | date = June 2014 | pmid = 24906146 | pmc = 4343198 | doi = 10.1016/j.cell.2014.05.010 }}</ref><ref name="Cong2013">{{cite journal | vauthors = Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F | author-link11=Feng Zhang | title = Multiplex genome engineering using CRISPR/Cas systems | journal = Science | volume = 339 | issue = 6121 | pages = 819–823 | date = February 2013 | pmid = 23287718 | pmc = 3795411 | doi = 10.1126/science.1231143 | bibcode = 2013Sci...339..819C }}</ref><ref name="Mali2013">{{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 = 823–826 | date = February 2013 | pmid = 23287722 | pmc = 3712628 | doi = 10.1126/science.1232033 | bibcode = 2013Sci...339..823M }}</ref>

=== Cas12a ===
{{main|Cas12a}}
Cas12a, a Class II Type V CRISPR-associated nuclease, was characterized in 2015 and was formerly known as Cpf1.<ref name="Rath_2015" /> This nuclease is found in the CRISPR-Cpf1 system of bacteria such as ''Francisella novicida''.<ref name="Redman_2016">{{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 = 213–215 | date = August 2016 | pmid = 27059283 | pmc = 4975809 | doi = 10.1136/archdischild-2016-310459 }}</ref><ref name="Horvath_2010">{{cite journal | vauthors = Horvath P, Barrangou R | author-link2=Rodolphe Barrangou | title = CRISPR/Cas, the immune system of bacteria and archaea | journal = Science | volume = 327 | issue = 5962 | pages = 167–170 | date = January 2010 | pmid = 20056882 | doi = 10.1126/science.1179555 | bibcode=2010Sci...327..167H }}</ref> The initial designation, derived from a TIGRFAMs protein family definition established in 2012, reflected the prevalence of this CRISPR-Cas subtype in the ''Prevotella'' and ''Francisella'' lineages. Cas12a exhibits several key distinctions from Cas9: it generates staggered cuts in double-stranded DNA, in contrast to the blunt ends produced by Cas9;<ref name="Proteintech">CRISPR-CAS9, TALENS and ZFNS – the battle in gene editing https://www.ptglab.com/news/blog/crispr-cas9-talens-and-zfns-the-battle-in-gene-editing/ {{Webarchive|url=https://web.archive.org/web/20210525031855/https://www.ptglab.com/news/blog/crispr-cas9-talens-and-zfns-the-battle-in-gene-editing/ |date=2021-05-25 }}</ref> it relies on a 'T-rich' protospacer adjacent motif (PAM) (typically 5'-TTTV-3', where V is A, C, or G), offering alternative targeting sites compared to the 'G-rich' PAMs (typically 5'-NGG-3') favored by Cas9;<ref name="Nobel_Foundation">{{cite web |title=Press release: The Nobel Prize in Chemistry 2020 |url=https://www.nobelprize.org/prizes/chemistry/2020/press-release/ |publisher=Nobel Foundation |access-date=7 October 2020 |archive-date=15 January 2021 |archive-url=https://web.archive.org/web/20210115172618/https://www.nobelprize.org/prizes/chemistry/2020/press-release/ |url-status=live }}</ref> and it requires only a CRISPR RNA (crRNA) for effective targeting, whereas Cas9 necessitates both a crRNA and a ''trans''-activating crRNA (tracrRNA).<ref name="Rath_2015">{{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 | pages = 119–128 | date = October 2015 | pmid = 25868999 | doi = 10.1016/j.biochi.2015.03.025 | doi-access = free }}</ref>

=== Cas13a ===
{{main| Cas13a}}
In 2016, the nuclease (formerly known as C2c2) from the bacterium ''Leptotrichia shahii'' was characterized by researchers in [[Feng Zhang]]'s group at [[MIT]] and the [[Broad Institute]]. Cas13 is an RNA-guided RNA endonuclease, which means that it does not cleave DNA, but only single-stranded RNA. Cas13 is guided by its crRNA to a ssRNA target and binds and cleaves the target. Similar to Cas12a, the Cas13 remains bound to the target and then cleaves other ssRNA molecules non-discriminately.<ref>{{cite journal | vauthors = Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F | title = C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector | journal = Science | volume = 353 | issue = 6299 | pages = aaf5573 | date = August 2016 | pmid = 27256883 | pmc = 5127784 | doi = 10.1126/science.aaf5573 }}</ref> This collateral cleavage property has been exploited for the development of various diagnostic technologies.<ref name="Nucleic acid detection with CRISPR">{{cite journal | vauthors = Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F | title = Nucleic acid detection with CRISPR-Cas13a/C2c2 | journal = Science | volume = 356 | issue = 6336 | pages = 438–442 | date = April 2017 | pmid = 28408723 | pmc = 5526198 | doi = 10.1126/science.aam9321 | bibcode = 2017Sci...356..438G }}</ref><ref>{{cite journal | vauthors = Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F | title = Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 | journal = Science | volume = 360 | issue = 6387 | pages = 439–444 | date = April 2018 | pmid = 29449508 | pmc = 5961727 | doi = 10.1126/science.aaq0179 | bibcode = 2018Sci...360..439G }}</ref><ref name="SPRINT: a Cas13a-based platform for">{{cite journal | vauthors = Iwasaki RS, Batey RT | title = SPRINT: a Cas13a-based platform for detection of small molecules | journal = Nucleic Acids Research | volume = 48 | issue = 17 | pages = e101 | date = September 2020 | pmid = 32797156 | pmc = 7515716 | doi = 10.1093/nar/gkaa673 | doi-access = free }}</ref>

== Locus structure ==
=== Repeats and spacers ===
The CRISPR array is made up of an AT-rich leader sequence followed by short repeats that are separated by unique spacers.<ref name="Hille2016">{{cite journal | vauthors = Hille F, Charpentier E | title = CRISPR-Cas: biology, mechanisms and relevance | journal = Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences | volume = 371 | issue = 1707 | date = November 2016 | pmid = 27672148 | pmc = 5052741 | doi = 10.1098/rstb.2015.0496 | page=20150496}}</ref> CRISPR repeats typically range in size from 28 to 37 [[base pair]]s (bps), though there can be as few as 23 bp and as many as 55 bp.<ref name="Barrangou2014">{{cite journal | vauthors = Barrangou R, Marraffini LA | author-link1=Rodolphe Barrangou | title = CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity | journal = Molecular Cell | volume = 54 | issue = 2 | pages = 234–244 | date = April 2014 | pmid = 24766887 | pmc = 4025954 | doi = 10.1016/j.molcel.2014.03.011 }}</ref> Some show [[dyad symmetry]], implying the formation of a [[nucleic acid secondary structure|secondary structure]] such as a [[stem-loop]] ('hairpin') in the RNA, while others are designed to be unstructured. The size of spacers in different CRISPR arrays is typically 32 to 38 bp (range 21 to 72 bp).<ref name="Barrangou2014"/> New spacers can appear rapidly as part of the immune response to phage infection.<ref name="pmid17894817"/> There are usually fewer than 50 units of the repeat-spacer sequence in a CRISPR array.<ref name="Barrangou2014"/>

=== CRISPR RNA structures ===
<gallery title="Gallery of secondary structure images" perrow="5">
Image:RF01315.png| CRISPR-DR2: Secondary structure taken from the [http://rfam.xfam.org Rfam] database. Family [http://rfam.xfam.org/family/RF01315 RF01315].
Image:RF01318.png| CRISPR-DR5: Secondary structure taken from the [http://rfam.xfam.org Rfam] database. Family [http://rfam.xfam.org/family/RF01318 RF011318].
Image:RF01319.png| CRISPR-DR6: Secondary structure taken from the [http://rfam.xfam.org Rfam] database. Family [http://rfam.xfam.org/family/RF01319 RF01319].
Image:RF01321.png| CRISPR-DR8: Secondary structure taken from the [http://rfam.xfam.org Rfam] database. Family [http://rfam.xfam.org/family/RF01321 RF01321].
Image:RF01322.png| CRISPR-DR9: Secondary structure taken from the [http://rfam.xfam.org Rfam] database. Family [http://rfam.xfam.org/family/RF01322 RF01322].
Image:RF01332.png| CRISPR-DR19: Secondary structure taken from the [http://rfam.xfam.org Rfam] database. Family [http://rfam.xfam.org/family/RF01332 RF01332].
Image:RF01350.png| CRISPR-DR41: Secondary structure taken from the [http://rfam.xfam.org Rfam] database. Family [http://rfam.xfam.org/family/RF01350 RF01350].
Image:RF01365.png| CRISPR-DR52: Secondary structure taken from the [http://rfam.xfam.org Rfam] database. Family [http://rfam.xfam.org/family/RF01365 RF01365].
Image:RF01370.png| CRISPR-DR57: Secondary structure taken from the [http://rfam.xfam.org Rfam] database. Family [http://rfam.xfam.org/family/RF01370 RF01370].
Image:RF01378.png| CRISPR-DR65: Secondary structure taken from the [http://rfam.xfam.org Rfam] database. Family [http://rfam.xfam.org/family/RF01378 RF01378].
</gallery>

=== Cas genes and CRISPR subtypes ===
Small clusters of ''cas'' genes are often located next to CRISPR repeat-spacer arrays. Collectively the 93 ''cas'' genes are grouped into 35 families based on sequence similarity of the encoded proteins. 11 of the 35 families form the ''cas'' core, which includes the protein families Cas1 through Cas9. A complete CRISPR-Cas locus has at least one gene belonging to the ''cas'' core.<!-- The numbers are from Makarova2015, and the Makarova2018 source has expanded them a bit. Its fig1 mentions 13 core gene families (instead of 11), but there's no mention of total gene families nor total gene count. --><ref name="Makarova2018">{{cite journal | vauthors = Koonin EV, Makarova KS | title = Origins and evolution of CRISPR-Cas systems | journal = Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences | volume = 374 | issue = 1772 | page = 20180087 | date = May 2019 | pmid = 30905284 | pmc = 6452270 | doi = 10.1098/rstb.2018.0087 | doi-access = free }}</ref>

CRISPR-Cas systems fall into two classes. Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV; class 2 is divided into types II, V, and VI.<ref name="Wright2016">{{cite journal | vauthors = Wright AV, Nuñez JK, Doudna JA | author-link3=Jennifer Doudna | title = Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering | journal = Cell | volume = 164 | issue = 1–2 | pages = 29–44 | date = January 2016 | pmid = 26771484 | doi = 10.1016/j.cell.2015.12.035 | doi-access = free }}</ref> The 6 system types are divided into 33 subtypes.<ref name="Makarova2020">{{cite journal |vauthors=Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJ, Charpentier E, Cheng D, Haft DH, Horvath P, Moineau S, Mojica FJ, Scott D, Shah SA, Siksnys V, Terns MP, Venclovas Č, White MF, Yakunin AF, Yan W, Zhang F, Garrett RA, Backofen R, van der Oost J, Barrangou R, Koonin EV | title = Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants | journal = Nature Reviews Microbiology | date = December 2019 | volume = 18 | issue = 1 |pages = 67–83 | doi = 10.1038/s41579-019-0299-x | doi-access = free | pmid = 31857715 | pmc = 8905525 | hdl = 10045/102627 | hdl-access = free }}</ref> Each type and most subtypes are characterized by a "signature gene" found almost exclusively in the category. Classification is also based on the complement of ''cas'' genes that are present. Most CRISPR-Cas systems have a Cas1 protein. The [[phylogeny]] of Cas1 proteins generally agrees with the classification system,<ref name="Makarova2015">{{cite journal | vauthors = Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV | author-link7=Rodolphe Barrangou | author-link21=Eugene Koonin | title = An updated evolutionary classification of CRISPR-Cas systems | journal = Nature Reviews. Microbiology | volume = 13 | issue = 11 | pages = 722–736 | date = November 2015 | pmid = 26411297 | pmc = 5426118 | doi = 10.1038/nrmicro3569 }}</ref> but exceptions exist due to module shuffling.<ref name="Makarova2018"/> Many organisms contain multiple CRISPR-Cas systems suggesting that they are compatible and may share components.<ref name="pmid22337052">{{cite journal | vauthors = Wiedenheft B, Sternberg SH, Doudna JA | author-link3=Jennifer Doudna | title = RNA-guided genetic silencing systems in bacteria and archaea | journal = Nature | volume = 482 | issue = 7385 | pages = 331–338 | date = February 2012 | pmid = 22337052 | doi = 10.1038/nature10886 | bibcode = 2012Natur.482..331W }}</ref><ref name="pmid23320564">{{cite journal | vauthors = Deng L, Garrett RA, Shah SA, Peng X, She Q | title = A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus | journal = Molecular Microbiology | volume = 87 | issue = 5 | pages = 1088–1099 | date = March 2013 | pmid = 23320564 | doi = 10.1111/mmi.12152 | doi-access =  }}</ref> The sporadic distribution of the CRISPR-Cas subtypes suggests that the CRISPR-Cas system is subject to [[horizontal gene transfer]] during microbial [[evolution]].

{{missing information|table|UniProt and InterPro cross-reference|date=October 2020}}
{| class="wikitable center" style="width:100%"
|+ Signature genes and their putative functions for the major and minor CRISPR-cas types
|-
! Class !! Cas type 
!Cas subtype!! Signature protein !! Function !! {{Refh}}
|-
| rowspan="19" | 1 || rowspan="8" | I
| {{sdash}} ||[[Cas3]] || Single-stranded DNA nuclease (HD domain) and ATP-dependent helicase ||<ref name="pmid21343909">{{cite journal | vauthors = Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V | author-link4=Rodolphe Barrangou | title = Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system | journal = The EMBO Journal | volume = 30 | issue = 7 | pages = 1335–1342 | date = April 2011 | pmid = 21343909 | pmc = 3094125 | doi = 10.1038/emboj.2011.41 }}</ref><ref name="ReferenceB">{{cite journal | vauthors = Huo Y, Nam KH, Ding F, Lee H, Wu L, Xiao Y, Farchione MD, Zhou S, Rajashankar K, Kurinov I, Zhang R, Ke A | title = Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation | journal = Nature Structural & Molecular Biology | volume = 21 | issue = 9 | pages = 771–777 | date = September 2014 | pmid = 25132177 | pmc = 4156918 | doi = 10.1038/nsmb.2875 }}</ref>
|-
|{{nowrap|I-A}}|| Cas8a, Cas5 || rowspan="3" | Cas8 is a Subunit of the interference module that is important in targeting of invading DNA by recognizing the [[Protospacer adjacent motif|PAM]] sequence. Cas5 is required for processing and stability of crRNAs. || rowspan="3" |<ref name="Makarova2015"/><ref>{{cite journal | vauthors = Brendel J, Stoll B, Lange SJ, Sharma K, Lenz C, Stachler AE, Maier LK, Richter H, Nickel L, Schmitz RA, Randau L, Allers T, Urlaub H, Backofen R, Marchfelder A | title = A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (crispr)-derived rnas (crrnas) in Haloferax volcanii | journal = The Journal of Biological Chemistry | volume = 289 | issue = 10 | pages = 7164–77 | date = March 2014 | pmid = 24459147 | doi = 10.1074/jbc.M113.508184 | pmc = 3945376 | doi-access = free }}</ref>
|-
|{{nowrap|I-B}}|| Cas8b
|-
|{{nowrap|I-C}}|| Cas8c
|-
|{{nowrap|I-D}}|| Cas10d || rowspan="2" | contains a domain homologous to the palm domain of nucleic acid polymerases and nucleotide cyclases || rowspan="2" |<ref name="pmid24728998">{{cite journal | vauthors = Chylinski K, Makarova KS, Charpentier E, Koonin EV | author-link4=Eugene Koonin | title = Classification and evolution of type II CRISPR-Cas systems | journal = Nucleic Acids Research | volume = 42 | issue = 10 | pages = 6091–6105 | date = June 2014 | pmid = 24728998 | pmc = 4041416 | doi = 10.1093/nar/gku241 }}</ref><ref name="pmid21756346">{{cite journal | vauthors = Makarova KS, Aravind L, Wolf YI, Koonin EV | author-link4=Eugene Koonin | title = Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems | journal = Biology Direct | volume = 6 | page = 38 | date = July 2011 | pmid = 21756346 | pmc = 3150331 | doi = 10.1186/1745-6150-6-38 | doi-access=free }}</ref>
|-
|{{nowrap|I-E}}|| Cse1, Cse2
|-
|{{nowrap|I-F}}|| Csy1, Csy2, Csy3 || Type IF-3 have been implicated in [[CRISPR-associated transposons]]||<ref name="Makarova2015"/>
|-
|{{nowrap|I-G}}{{#tag:ref | Subtype {{nowrap|I-G}} was previously known as subtype {{nowrap|I-U}}.<ref name="Makarova2015"/> | group = Note | name = {{nowrap|I-U}} }}|| GSU0054 || ||<ref name="Makarova2019">{{cite journal | vauthors = Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJ, Charpentier E, Cheng D, Haft DH, Horvath P, Moineau S, Mojica FJ, Scott D, Shah SA, Siksnys V, Terns MP, Venclovas Č, White MF, Yakunin AF, Yan W, Zhang F, Garrett RA, Backofen R, van der Oost J, Barrangou R, Koonin EV | title = Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants | journal = Nature Reviews. Microbiology | volume = 18 | issue = 2 | pages = 67–83 | date = February 2020 | pmid = 31857715 | doi = 10.1038/s41579-019-0299-x | pmc = 8905525 | hdl-access = free | hdl = 10045/102627 }}</ref>
|-
| rowspan="7" | III
| {{sdash}} || Cas10 || [[Homologous series|Homolog]] of Cas10d and Cse1. Binds CRISPR target RNA and promotes stability of the interference complex ||<ref name="pmid21756346"/><ref name="pmid30840895">{{cite journal | vauthors = Mogila I, Kazlauskiene M, Valinskyte S, Tamulaitiene G, Tamulaitis G, Siksnys V | title = Genetic Dissection of the Type {{nowrap|III-A}} CRISPR-Cas System Csm Complex Reveals Roles of Individual Subunits | journal = Cell Reports | volume = 26 | issue = 10 | pages = 2753–2765.e4 | date = March 2019 | pmid = 30840895 | doi = 10.1016/j.celrep.2019.02.029 | doi-access = free }}</ref>
|-
|{{nowrap|III-A}}|| Csm2 || Not determined ||<ref name="Makarova2015"/>
|-
|{{nowrap|III-B}}|| Cmr5 || Not determined ||<ref name="Makarova2015"/>
|-
|{{nowrap|III-C}}|| Cas10 or Csx11 || ||<ref name="Makarova2015"/><ref name="pmid30840895"/>
|-
|{{nowrap|III-D}}|| Csx10 || ||<ref name="Makarova2015"/>
|-
|{{nowrap|III-E}}|| || ||<ref name="Makarova2019"/>
|-
|{{nowrap|III-F}}|| || ||<ref name="Makarova2019"/>
|-
| rowspan="4" | IV
| {{sdash}} || Csf1 || ||<ref name="Makarova2019"/>
|-
|{{nowrap|IV-A}}|| || ||<ref name="Makarova2019"/>
|-
|{{nowrap|IV-B}}|| || ||<ref name="Makarova2019"/>
|-
|{{nowrap|IV-C}}|| || ||<ref name="Makarova2019"/>
|-
| rowspan="23" | 2 || rowspan="4" | II
| {{sdash}} ||[[Cas9]] || [[Nuclease]]s RuvC and HNH together produce [[double-strand breaks|DSBs]], and separately can produce single-strand breaks. Ensures the acquisition of functional spacers during adaptation. ||<ref name="pmid22949671">{{cite journal | vauthors = Gasiunas G, Barrangou R, Horvath P, Siksnys V | author-link2=Rodolphe Barrangou | title = Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 39 | pages = E2579–2586 | date = September 2012 | pmid = 22949671 | pmc = 3465414 | doi = 10.1073/pnas.1208507109 | bibcode = 2012PNAS..109E2579G | doi-access=free }}</ref><ref>{{cite journal | vauthors = Heler R, Samai P, Modell JW, Weiner C, Goldberg GW, Bikard D, Marraffini LA | title = Cas9 specifies functional viral targets during CRISPR-Cas adaptation | journal = Nature | volume = 519 | issue = 7542 | pages = 199–202 | date = March 2015 | pmid = 25707807 | pmc = 4385744 | doi = 10.1038/nature14245 | bibcode = 2015Natur.519..199H }}</ref>
|-
|{{nowrap|II-A}}|| Csn2 || Ring-shaped DNA-binding protein. Involved in primed adaptation in Type II CRISPR system. ||<ref>{{cite journal | vauthors = Nam KH, Kurinov I, Ke A | title = Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity | journal = The Journal of Biological Chemistry | volume = 286 | issue = 35 | pages = 30759–30768 | date = September 2011 | pmid = 21697083 | pmc = 3162437 | doi = 10.1074/jbc.M111.256263 | doi-access = free }}</ref>
|-
|{{nowrap|II-B}}||[[Cas4]] || Endonuclease that works with cas1 and cas2 to generate spacer sequences || <ref>{{cite journal | vauthors = Lee H, Dhingra Y, Sashital DG | title = The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation | journal = eLife | volume = 8 | date = April 2019 | pmid = 31021314 | doi = 10.7554/eLife.44248 | pmc = 6519985 | doi-access = free }}</ref>
|-
|{{nowrap|II-C}}|| || Characterized by the absence of either Csn2 or Cas4 ||<ref name="pmid23563642">{{cite journal | vauthors = Chylinski K, Le Rhun A, Charpentier E | title = The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems | journal = RNA Biology | volume = 10 | issue = 5 | pages = 726–737 | date = May 2013 | pmid = 23563642 | pmc = 3737331 | doi = 10.4161/rna.24321 }}</ref>
|-
| rowspan="12" | V
| {{sdash}} || Cas12 || Nuclease RuvC. Lacks HNH. ||<ref name="Wright2016"/><ref>{{cite journal | vauthors = Makarova KS, Zhang F, Koonin EV | title = SnapShot: Class 2 CRISPR-Cas Systems | journal = Cell | volume = 168 | issue = 1–2 | pages = 328–328.e1 | date = January 2017 | pmid = 28086097 | doi = 10.1016/j.cell.2016.12.038 | doi-access = free }}</ref>
|-
|{{nowrap|V-A}}|| [[Cas12a]] (Cpf1) || Auto-processing pre-crRNA activity for multiplex gene regulation||<ref name="Makarova2019"/><ref>{{cite journal | vauthors = Paul B, Montoya G | title = CRISPR-Cas12a: Functional overview and applications | journal = Biomedical Journal | volume = 43 | issue = 1 | pages = 8–17 | date = February 2020 | pmid = 32200959 | pmc = 7090318 | doi = 10.1016/j.bj.2019.10.005 }}</ref>
|-
|{{nowrap|V-B}}|| Cas12b (C2c1) || ||<ref name="Makarova2019"/>
|-
|{{nowrap|V-C}}|| Cas12c (C2c3) || ||<ref name="Makarova2019"/>
|-
|{{nowrap|V-D}}|| Cas12d (CasY) || ||<ref name="Makarova2019"/>
|-
|{{nowrap|V-E}}|| Cas12e (CasX) || ||<ref name="Makarova2019"/>
|-
|{{nowrap|V-F}}|| Cas12f (Cas14, C2c10) || ||<ref name="Makarova2019"/>
|-
|{{nowrap|V-G}}|| Cas12g || ||<ref name="Makarova2019"/>
|-
|{{nowrap|V-H}}|| Cas12h || ||<ref name="Makarova2019"/>
|-
|{{nowrap|V-I}}|| Cas12i || ||<ref name="Makarova2019"/>
|-
|{{nowrap|V-K}}{{#tag:ref | Subtype {{nowrap|V-K}} was previously known as subtype {{nowrap|V-U}}5.<ref name="Makarova2019"/> | group = Note | name = {{nowrap|V-U}}5 }}|| Cas12k (C2c5) || Type V-K have been implicated in [[CRISPR-associated transposons]].||<ref name="Makarova2019"/>
|-
|{{nowrap|V-U}}|| C2c4, C2c8, C2c9 || ||<ref name="Makarova2019"/>
|-
| rowspan="7" | VI
| {{sdash}} || Cas13 || RNA-guided RNase || <ref name="Wright2016"/><ref>{{cite journal | vauthors = Cox DB, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F | author-link7=Feng Zhang | title = RNA editing with CRISPR-Cas13 | journal = Science | volume = 358 | issue = 6366 | pages = 1019–1027 | date = November 2017 | pmid = 29070703 | pmc = 5793859 | doi = 10.1126/science.aaq0180 | bibcode = 2017Sci...358.1019C }}</ref>
|-
|{{nowrap|VI-A}}|| Cas13a (C2c2) || ||<ref name="Makarova2019"/>
|-
|{{nowrap|VI-B}}|| Cas13b || ||<ref name="Makarova2019"/>
|-
|{{nowrap|VI-C}}|| Cas13c || ||<ref name="Makarova2019"/>
|-
|{{nowrap|VI-D}}|| Cas13d || ||<ref name="Makarova2019"/>
|-
|VI-X
|Cas13x.1
|RNA dependent RNA polymerase, Prophylactic RNA-virus inhibition
|<ref name="Xu-2021">{{cite journal | vauthors = Xu C, Zhou Y, Xiao Q, He B, Geng G, Wang Z, Cao B, Dong X, Bai W, Wang Y, Wang X, Zhou D, Yuan T, Huo X, Lai J, Yang H | title = Programmable RNA editing with compact CRISPR-Cas13 systems from uncultivated microbes | journal = Nature Methods | volume = 18 | issue = 5 | pages = 499–506 | date = May 2021 | pmid = 33941935 | doi = 10.1038/s41592-021-01124-4 | doi-access = free }}</ref>
|-
|VI-Y
|
|
|<ref name="Xu-2021"/>
|}
{{Clear}}

== Mechanism ==
[[File:The Stages of CRISPR immunity.svg|thumb|The stages of CRISPR immunity for each of the 3 major types of adaptive immunity.<br/> (1) Acquisition begins by recognition of invading DNA by [[Cas1]] and Cas2 and cleavage of a protospacer.<br/> (2) The protospacer is ligated to the direct repeat adjacent to the leader sequence and <br/>(3) single strand extension repairs the CRISPR and duplicates the direct repeat. The crRNA processing and interference stages occur differently in each of the three major CRISPR systems.<br/> (4) The primary CRISPR transcript is cleaved by cas genes to produce crRNAs.<br/> (5) In type I systems Cas6e/Cas6f cleave at the junction of ssRNA and dsRNA formed by hairpin loops in the direct repeat. Type II systems use a trans-activating (tracr) RNA to form dsRNA, which is cleaved by [[Cas9]] and RNaseIII. Type III systems use a Cas6 homolog that does not require hairpin loops in the direct repeat for cleavage.<br/> (6) In type II and type III systems secondary trimming is performed at either the 5' or 3' end to produce mature crRNAs.<br/> (7) Mature crRNAs associate with Cas proteins to form interference complexes.<br/> (8) In type I and type II systems, interactions between the protein and PAM sequence are required for degradation of invading DNA. Type III systems do not require a PAM for successful degradation and in type III-A systems basepairing occurs between the crRNA and mRNA rather than the DNA, targeted by type III-B systems.]]
[[File:12 Hegasy Cas9 Immun Wiki E CCBYSA.png|thumb|The CRISPR genetic locus provides bacteria with a defense mechanism to protect them from repeated phage infections.]]
[[File:13 Hegasy CRISPR pre crRNA Wiki E CCBYSA.png|thumb|Transcripts of the CRISPR Genetic Locus and Maturation of pre-crRNA]]
[[File:14 Hegasy Cas9 3D Complex Wiki E CCBYSA.png|thumb|3D structure of the CRISPR-Cas9 interference complex]]
[[File:15 Hegasy Cas9 DNA Tool Wiki E CCBYSA.png|thumb|CRISPR-Cas9 as a molecular tool introduces targeted double strand DNA breaks.]]
[[File:16 Hegasy DNA Rep Wiki E CCBYSA.png|thumb|Double-strand DNA breaks introduced by CRISPR-Cas9 allows further genetic manipulation by exploiting endogenous DNA repair mechanisms.]]

CRISPR-Cas immunity is a natural process of bacteria and archaea.<ref name="pmid33117331">{{cite journal | vauthors = Azangou-Khyavy M, Ghasemi M, Khanali J, Boroomand-Saboor M, Jamalkhah M, Soleimani M, Kiani J | title = CRISPR/Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer | journal = Frontiers in Immunology | volume = 11 | issue = | page = 2062 | date = 2020 | pmid = 33117331 | pmc = 7553049 | doi = 10.3389/fimmu.2020.02062 | doi-access = free }}</ref> CRISPR-Cas prevents bacteriophage infection, [[Bacterial conjugation|conjugation]] and [[transformation (genetics)#Natural transformation|natural transformation]] by degrading foreign nucleic acids that enter the cell.<ref name="Marraffini2015">{{cite journal | vauthors = Marraffini LA | title = CRISPR-Cas immunity in prokaryotes | journal = Nature | volume = 526 | issue = 7571 | pages = 55–61 | date = October 2015 | pmid = 26432244 | doi = 10.1038/nature15386 | bibcode = 2015Natur.526...55M }}</ref>

=== Spacer acquisition ===
When a [[microbe]] is invaded by a [[bacteriophage]], the first stage of the immune response is to capture phage DNA and insert it into a CRISPR locus in the form of a spacer. [[Cas1]] and [[Cas2]] are found in both types of CRISPR-Cas immune systems, which indicates that they are involved in spacer acquisition. Mutation studies confirmed this hypothesis, showing that removal of Cas1 or Cas2 stopped spacer acquisition, without affecting CRISPR immune response.<ref name="pmid19120484">{{cite journal | vauthors = Aliyari R, Ding SW | title = RNA-based viral immunity initiated by the Dicer family of host immune receptors | journal = Immunological Reviews | volume = 227 | issue = 1 | pages = 176–188 | date = January 2009 | pmid = 19120484 | pmc = 2676720 | doi = 10.1111/j.1600-065X.2008.00722.x }}</ref><ref name="pmid23696746">{{cite journal | vauthors = Dugar G, Herbig A, Förstner KU, Heidrich N, Reinhardt R, Nieselt K, Sharma CM | title = High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni isolates | journal = PLOS Genetics | volume = 9 | issue = 5 | pages = e1003495 | date = May 2013 | pmid = 23696746 | pmc = 3656092 | doi = 10.1371/journal.pgen.1003495 | doi-access = free }}</ref><ref name="pmid22160698">{{cite journal | vauthors = Hatoum-Aslan A, Maniv I, Marraffini LA | title = Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 108 | issue = 52 | pages = 21218–21222 | date = December 2011 | pmid = 22160698 | pmc = 3248500 | doi = 10.1073/pnas.1112832108 | bibcode = 2011PNAS..10821218H | doi-access = free }}</ref><ref name="pmid22402487">{{cite journal | vauthors = Yosef I, Goren MG, Qimron U | title = Proteins and DNA elements essential for the CRISPR adaptation process in ''Escherichia coli'' | journal = Nucleic Acids Research | volume = 40 | issue = 12 | pages = 5569–5576 | date = July 2012 | pmid = 22402487 | pmc = 3384332 | doi = 10.1093/nar/gks216 }}</ref><ref name="pmid22558257">{{cite journal | vauthors = Swarts DC, Mosterd C, van Passel MW, Brouns SJ | title = CRISPR interference directs strand specific spacer acquisition | journal = PLOS ONE | volume = 7 | issue = 4 | pages = e35888 | year = 2012 | pmid = 22558257 | pmc = 3338789 | doi = 10.1371/journal.pone.0035888 | bibcode = 2012PLoSO...735888S | doi-access = free }}</ref>

Multiple Cas1 proteins have been characterised and their structures resolved.<ref name="pmid21219465">{{cite journal | vauthors = Babu M, Beloglazova N, Flick R, Graham C, Skarina T, Nocek B, Gagarinova A, Pogoutse O, Brown G, Binkowski A, Phanse S, Joachimiak A, Koonin EV, Savchenko A, Emili A, Greenblatt J, Edwards AM, Yakunin AF | author-link=Eugene Koonin | title = A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair | journal = Molecular Microbiology | volume = 79 | issue = 2 | pages = 484–502 | date = January 2011 | pmid = 21219465 | pmc = 3071548 | doi = 10.1111/j.1365-2958.2010.07465.x }}</ref><ref name="pmid19427858">{{cite journal | vauthors = Han D, Lehmann K, Krauss G | title = SSO1450—a CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA | journal = FEBS Letters | volume = 583 | issue = 12 | pages = 1928–1932 | date = June 2009 | pmid = 19427858 | doi = 10.1016/j.febslet.2009.04.047 | doi-access =  | bibcode = 2009FEBSL.583.1928H }}</ref><ref name="pmid19523907">{{cite journal | vauthors = Wiedenheft B, Zhou K, Jinek M, Coyle SM, Ma W, Doudna JA | author-link6=Jennifer Doudna | title = Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense | journal = Structure | volume = 17 | issue = 6 | pages = 904–912 | date = June 2009 | pmid = 19523907 | doi = 10.1016/j.str.2009.03.019 | doi-access = free }}</ref> Cas1 proteins have diverse [[amino acid]] sequences. However, their crystal structures are similar and all purified Cas1 proteins are metal-dependent nucleases/[[integrases]] that bind to DNA in a sequence-independent manner.<ref name="pmid22337052"/> Representative Cas2 proteins have been characterised and possess either (single strand) ssRNA-<ref name="pmid18482976">{{cite journal | vauthors = Beloglazova N, Brown G, Zimmerman MD, Proudfoot M, Makarova KS, Kudritska M, Kochinyan S, Wang S, Chruszcz M, Minor W, Koonin EV, Edwards AM, Savchenko A, Yakunin AF | author-link11=Eugene Koonin | title = A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats | journal = The Journal of Biological Chemistry | volume = 283 | issue = 29 | pages = 20361–20371 | date = July 2008 | pmid = 18482976 | pmc = 2459268 | doi = 10.1074/jbc.M803225200 | doi-access=free }}</ref> or (double strand) dsDNA-<ref name="pmid21139194">{{cite journal | vauthors = Samai P, Smith P, Shuman S | title = Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris | journal = Acta Crystallographica Section F | volume = 66 | issue = Pt 12 | pages = 1552–1556 | date = December 2010 | pmid = 21139194 | pmc = 2998353 | doi = 10.1107/S1744309110039801 }}</ref><ref name="pmid22942283">{{cite journal | vauthors = Nam KH, Ding F, Haitjema C, Huang Q, DeLisa MP, Ke A | title = Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein | journal = The Journal of Biological Chemistry | volume = 287 | issue = 43 | pages = 35943–35952 | date = October 2012 | pmid = 22942283 | pmc = 3476262 | doi = 10.1074/jbc.M112.382598 | doi-access = free }}</ref> specific [[endoribonuclease]] activity.

In the I-E system of ''E. coli'', Cas1 and Cas2 form a complex where a Cas2 dimer bridges two Cas1 dimers.<ref name="pmid24793649">{{cite journal | vauthors = Nuñez JK, Kranzusch PJ, Noeske J, Wright AV, Davies CW, Doudna JA | author-link6=Jennifer Doudna | title = Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity | journal = Nature Structural & Molecular Biology | volume = 21 | issue = 6 | pages = 528–534 | date = June 2014 | pmid = 24793649 | pmc = 4075942 | doi = 10.1038/nsmb.2820 }}</ref> In this complex, Cas2 performs a non-enzymatic scaffolding role,<ref name="pmid24793649"/> binding double-stranded fragments of invading DNA, while Cas1 binds the single-stranded flanks of the DNA and catalyses their integration into CRISPR arrays.<ref name="pmid25707795">{{cite journal | vauthors = Nuñez JK, Lee AS, Engelman A, Doudna JA | author-link4=Jennifer Doudna | title = Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity | journal = Nature | volume = 519 | issue = 7542 | pages = 193–198 | date = March 2015 | pmid = 25707795 | pmc = 4359072 | doi = 10.1038/nature14237 | bibcode = 2015Natur.519..193N }}</ref><ref name="pmid26478180">{{cite journal | vauthors = Wang J, Li J, Zhao H, Sheng G, Wang M, Yin M, Wang Y | title = Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems | journal = Cell | volume = 163 | issue = 4 | pages = 840–853 | date = November 2015 | pmid = 26478180 | doi = 10.1016/j.cell.2015.10.008 | doi-access = free }}</ref><ref name="pmid26503043">{{cite journal | vauthors = Nuñez JK, Harrington LB, Kranzusch PJ, Engelman AN, Doudna JA | author-link5=Jennifer Doudna | title = Foreign DNA capture during CRISPR-Cas adaptive immunity | journal = Nature | volume = 527 | issue = 7579 | pages = 535–538 | date = November 2015 | pmid = 26503043 | pmc = 4662619 | doi = 10.1038/nature15760 | bibcode = 2015Natur.527..535N }}</ref> New spacers are usually added at the beginning of the CRISPR next to the leader sequence creating a chronological record of viral infections.<ref>{{cite journal | vauthors = Sorek R, Lawrence CM, Wiedenheft B | title = CRISPR-mediated adaptive immune systems in bacteria and archaea | journal = Annual Review of Biochemistry | volume = 82 | issue = 1 | pages = 237–266 | year = 2013 | pmid = 23495939 | doi = 10.1146/annurev-biochem-072911-172315 | doi-access = free }}</ref> In ''E. coli'' a [[Bacterial DNA binding protein|histone like protein]] called integration host factor ([[Bacterial DNA binding protein|IHF]]), which binds to the leader sequence, is responsible for the accuracy of this integration.<ref>{{cite journal | vauthors = Nuñez JK, Bai L, Harrington LB, Hinder TL, Doudna JA | author-link5=Jennifer Doudna | title = CRISPR Immunological Memory Requires a Host Factor for Specificity | journal = Molecular Cell | volume = 62 | issue = 6 | pages = 824–833 | date = June 2016 | pmid = 27211867 | doi = 10.1016/j.molcel.2016.04.027 | doi-access = free }}</ref> IHF also enhances integration efficiency in the type I-F system of ''[[Pectobacterium atrosepticum]],''<ref>{{cite journal | vauthors = Fagerlund RD, Wilkinson ME, Klykov O, Barendregt A, Pearce FG, Kieper SN, Maxwell HW, Capolupo A, Heck AJ, Krause KL, Bostina M, Scheltema RA, Staals RH, Fineran PC | title = Spacer capture and integration by a type I-F Cas1-Cas2–3 CRISPR adaptation complex | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 114 | issue = 26 | pages = E5122–E5128 | date = June 2017 | pmid = 28611213 | pmc = 5495228 | doi = 10.1073/pnas.1618421114 | bibcode = 2017PNAS..114E5122F | doi-access = free }}</ref> but in other systems, different host factors may be required<ref>{{cite journal | vauthors = Rollie C, Graham S, Rouillon C, White MF | title = Prespacer processing and specific integration in a Type I-A CRISPR system | journal = Nucleic Acids Research | volume = 46 | issue = 3 | pages = 1007–1020 | date = February 2018 | pmid = 29228332 | pmc = 5815122 | doi = 10.1093/nar/gkx1232 }}</ref>

==== Protospacer adjacent motifs (PAM)====
{{Main|Protospacer adjacent motif}}
Bioinformatic analysis of regions of phage genomes that were excised as spacers (termed protospacers) revealed that they were not randomly selected but instead were found adjacent to short (3–5 bp) DNA sequences termed [[protospacer adjacent motif]]s (PAM). Analysis of CRISPR-Cas systems showed PAMs to be important for type I and type II, but not type III systems during acquisition.<ref name="pmid16079334"/><ref name="pmid18065539">{{cite journal | vauthors = Horvath P, Romero DA, Coûté-Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P, Fremaux C, Barrangou R | author-link9=Rodolphe Barrangou | title = Diversity, activity, and evolution of CRISPR loci in ''Streptococcus thermophilus'' | journal = Journal of Bacteriology | volume = 190 | issue = 4 | pages = 1401–1412 | date = February 2008 | pmid = 18065539 | pmc = 2238196 | doi = 10.1128/JB.01415-07 }}</ref><ref name="pmid18065545">{{cite journal | vauthors = Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C, Boyaval P, Romero DA, Horvath P, Moineau S | author-link2=Rodolphe Barrangou | title = Phage response to CRISPR-encoded resistance in ''Streptococcus thermophilus'' | journal = Journal of Bacteriology | volume = 190 | issue = 4 | pages = 1390–1400 | date = February 2008 | pmid = 18065545 | pmc = 2238228 | doi = 10.1128/JB.01412-07 }}</ref><ref name="pmid19246744">{{cite journal | vauthors = Mojica FJ, Díez-Villaseñor C, García-Martínez J, Almendros C | title = Short motif sequences determine the targets of the prokaryotic CRISPR defence system | journal = Microbiology | volume = 155 | issue = Pt 3 | pages = 733–740 | date = March 2009 | pmid = 19246744 | doi = 10.1099/mic.0.023960-0 | doi-access = free }}</ref><ref name="pmid19239620">{{cite journal | vauthors = Lillestøl RK, Shah SA, Brügger K, Redder P, Phan H, Christiansen J, Garrett RA | title = CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties | journal = Molecular Microbiology | volume = 72 | issue = 1 | pages = 259–272 | date = April 2009 | pmid = 19239620 | doi = 10.1111/j.1365-2958.2009.06641.x }}</ref><ref name="pmid19143596">{{cite journal | vauthors = Shah SA, Hansen NR, Garrett RA | title = Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism | journal = Biochemical Society Transactions | volume = 37 | issue = Pt 1 | pages = 23–28 | date = February 2009 | pmid = 19143596 | doi = 10.1042/BST0370023 }}</ref> In type I and type II systems, protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer cut using a ruler mechanism, thus maintaining the regularity of the spacer size in the CRISPR array.<ref name="pmid23445770">{{cite journal | vauthors = Díez-Villaseñor C, Guzmán NM, Almendros C, García-Martínez J, Mojica FJ | title = CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of ''Escherichia coli'' | journal = RNA Biology | volume = 10 | issue = 5 | pages = 792–802 | date = May 2013 | pmid = 23445770 | pmc = 3737337 | doi = 10.4161/rna.24023 }}</ref><ref name="pmid22834906">{{cite journal | vauthors = Erdmann S, Garrett RA | title = Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms | journal = Molecular Microbiology | volume = 85 | issue = 6 | pages = 1044–1056 | date = September 2012 | pmid = 22834906 | pmc = 3468723 | doi = 10.1111/j.1365-2958.2012.08171.x }}</ref> The conservation of the PAM sequence differs between CRISPR-Cas systems and appears to be evolutionarily linked to Cas1 and the [[Leader sequence (mRNA)|leader sequence]].<ref name="pmid19143596"/><ref name="pmid23403393">{{cite journal | vauthors = Shah SA, Erdmann S, Mojica FJ, Garrett RA | title = Protospacer recognition motifs: mixed identities and functional diversity | journal = RNA Biology | volume = 10 | issue = 5 | pages = 891–899 | date = May 2013 | pmid = 23403393 | pmc = 3737346 | doi = 10.4161/rna.23764 }}</ref>

New spacers are added to a CRISPR array in a directional manner,<ref name="pmid15758212"/> occurring preferentially,<ref name="pmid17894817">{{cite journal | vauthors = Tyson GW, Banfield JF | title = Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses | journal = Environmental Microbiology | volume = 10 | issue = 1 | pages = 200–207 | date = January 2008 | pmid = 17894817 | doi = 10.1111/j.1462-2920.2007.01444.x | bibcode = 2008EnvMi..10..200T }}</ref><ref name="pmid18065539"/><ref name="pmid18065545"/><ref name="pmid18497291">{{cite journal | vauthors = Andersson AF, Banfield JF | title = Virus population dynamics and acquired virus resistance in natural microbial communities | journal = Science | volume = 320 | issue = 5879 | pages = 1047–1050 | date = May 2008 | pmid = 18497291 | doi = 10.1126/science.1157358 | bibcode = 2008Sci...320.1047A }}</ref><ref name="pmid21149389">{{cite journal | vauthors = Pride DT, Sun CL, Salzman J, Rao N, Loomer P, Armitage GC, Banfield JF, Relman DA | title = Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time | journal = Genome Research | volume = 21 | issue = 1 | pages = 126–136 | date = January 2011 | pmid = 21149389 | pmc = 3012920 | doi = 10.1101/gr.111732.110 }}</ref> but not exclusively, adjacent<ref name="pmid19239620"/><ref name="pmid22834906"/> to the leader sequence. Analysis of the type I-E system from ''E. coli'' demonstrated that the first direct repeat adjacent to the leader sequence is copied, with the newly acquired spacer inserted between the first and second direct repeats.<ref name="pmid22402487"/><ref name="pmid23445770"/>

The PAM sequence appears to be important during spacer insertion in type I-E systems. That sequence contains a strongly conserved final nucleotide (nt) adjacent to the first nt of the protospacer. This nt becomes the final base in the first direct repeat.<ref name="pmid22558257"/><ref name="pmid22771574">{{cite journal | vauthors = Goren MG, Yosef I, Auster O, Qimron U | title = Experimental definition of a clustered regularly interspaced short palindromic duplicon in ''Escherichia coli'' | journal = Journal of Molecular Biology | volume = 423 | issue = 1 | pages = 14–16 | date = October 2012 | pmid = 22771574 | doi = 10.1016/j.jmb.2012.06.037 }}</ref><ref name="pmid22781758">{{cite journal | vauthors = Datsenko KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E | title = Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system | journal = Nature Communications | volume = 3 | page = 945 | date = July 2012 | pmid = 22781758 | doi = 10.1038/ncomms1937 | bibcode = 2012NatCo...3..945D | doi-access = free }}</ref> This suggests that the spacer acquisition machinery generates single-stranded overhangs in the second-to-last position of the direct repeat and in the PAM during spacer insertion. However, not all CRISPR-Cas systems appear to share this mechanism as PAMs in other organisms do not show the same level of conservation in the final position.<ref name="pmid23403393"/> It is likely that in those systems, a blunt end is generated at the very end of the direct repeat and the protospacer during acquisition.

==== Insertion variants ====
Analysis of ''[[Sulfolobus solfataricus]]'' CRISPRs revealed further complexities to the canonical model of spacer insertion, as one of its six CRISPR loci inserted new spacers randomly throughout its CRISPR array, as opposed to inserting closest to the leader sequence.<ref name="pmid22834906"/>

Multiple CRISPRs contain many spacers to the same phage. The mechanism that causes this phenomenon was discovered in the type I-E system of ''E. coli''. A significant enhancement in spacer acquisition was detected where spacers already target the phage, even mismatches to the protospacer. This 'priming' requires the Cas proteins involved in both acquisition and interference to interact with each other. Newly acquired spacers that result from the priming mechanism are always found on the same strand as the priming spacer.<ref name="pmid22558257"/><ref name="pmid22771574"/><ref name="pmid22781758"/> This observation led to the hypothesis that the acquisition machinery slides along the foreign DNA after priming to find a new protospacer.<ref name="pmid22781758"/>

=== Biogenesis ===
CRISPR-RNA (crRNA), which later guides the Cas nuclease to the target during the interference step, must be generated from the CRISPR sequence. The crRNA is initially transcribed as part of a single long transcript encompassing much of the CRISPR array.<ref name="pmid20125085"/> This transcript is then cleaved by Cas proteins to form crRNAs. The mechanism to produce crRNAs differs among CRISPR-Cas systems. In type I-E and type I-F systems, the proteins Cas6e and Cas6f respectively, recognise stem-loops<ref name="pmid21572444">{{cite journal | vauthors = Gesner EM, Schellenberg MJ, Garside EL, George MM, Macmillan AM | title = Recognition and maturation of effector RNAs in a CRISPR interference pathway | journal = Nature Structural & Molecular Biology | volume = 18 | issue = 6 | pages = 688–692 | date = June 2011 | pmid = 21572444 | doi = 10.1038/nsmb.2042 }}</ref><ref name="pmid21572442">{{cite journal | vauthors = Sashital DG, Jinek M, Doudna JA | author-link3=Jennifer Doudna | title = An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3 | journal = Nature Structural & Molecular Biology | volume = 18 | issue = 6 | pages = 680–687 | date = June 2011 | pmid = 21572442 | doi = 10.1038/nsmb.2043 }}</ref><ref name="pmid20829488">{{cite journal | vauthors = Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA | author-link5=Jennifer Doudna | title = Sequence- and structure-specific RNA processing by a CRISPR endonuclease | journal = Science | volume = 329 | issue = 5997 | pages = 1355–1358 | date = September 2010 | pmid = 20829488 | pmc = 3133607 | doi = 10.1126/science.1192272 | bibcode = 2010Sci...329.1355H }}</ref> created by the pairing of identical repeats that flank the crRNA.<ref name="pmid17442114">{{cite journal | vauthors = Kunin V, Sorek R, Hugenholtz P | title = Evolutionary conservation of sequence and secondary structures in CRISPR repeats | journal = Genome Biology | volume = 8 | issue = 4 | pages = R61 | year = 2007 | pmid = 17442114 | pmc = 1896005 | doi = 10.1186/gb-2007-8-4-r61 | doi-access = free }}</ref> These Cas proteins cleave the longer transcript at the edge of the paired region, leaving a single crRNA along with a small remnant of the paired repeat region.

Type III systems also use Cas6, however, their repeats do not produce stem-loops. Cleavage instead occurs by the longer transcript wrapping around the Cas6 to allow cleavage just upstream of the repeat sequence.<ref name="pmid19141480">{{cite journal | vauthors = Carte J, Wang R, Li H, Terns RM, Terns MP | title = Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes | journal = Genes & Development | volume = 22 | issue = 24 | pages = 3489–3496 | date = December 2008 | pmid = 19141480 | pmc = 2607076 | doi = 10.1101/gad.1742908 }}</ref><ref name="pmid21300293">{{cite journal | vauthors = Wang R, Preamplume G, Terns MP, Terns RM, Li H | title = Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage | journal = Structure | volume = 19 | issue = 2 | pages = 257–264 | date = February 2011 | pmid = 21300293 | pmc = 3154685 | doi = 10.1016/j.str.2010.11.014 }}</ref><ref name="pmid24150936">{{cite journal | vauthors = Niewoehner O, Jinek M, Doudna JA | author-link3=Jennifer Doudna | title = Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases | journal = Nucleic Acids Research | volume = 42 | issue = 2 | pages = 1341–1353 | date = January 2014 | pmid = 24150936 | pmc = 3902920 | doi = 10.1093/nar/gkt922 }}</ref>

Type II systems lack the Cas6 gene and instead utilize RNaseIII for cleavage. Functional type II systems encode an extra small RNA that is complementary to the repeat sequence, known as a [[trans-activating crRNA]] (tracrRNA).<ref name="Deltcheva2011"/> Transcription of the tracrRNA and the primary CRISPR transcript results in base pairing and the formation of dsRNA at the repeat sequence, which is subsequently targeted by RNaseIII to produce crRNAs. Unlike the other two systems, the crRNA does not contain the full spacer, which is instead truncated at one end.<ref name="pmid22949671"/>

CrRNAs associate with Cas proteins to form ribonucleotide complexes that recognize foreign nucleic acids. CrRNAs show no preference between the coding and non-coding strands, which is indicative of an RNA-guided DNA-targeting system.<ref name="pmid19095942"/><ref name="Garneau2010"/><ref name="pmid19120484"/><ref name="pmid22558257"/><ref name="pmid21646539">{{cite journal | vauthors = Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, Severinov K | title = Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 108 | issue = 25 | pages = 10098–10103 | date = June 2011 | pmid = 21646539 | pmc = 3121866 | doi = 10.1073/pnas.1104144108 | bibcode = 2011PNAS..10810098S | doi-access = free }}</ref><ref name="pmid21166892">{{cite journal | vauthors = Gudbergsdottir S, Deng L, Chen Z, Jensen JV, Jensen LR, She Q, Garrett RA | title = Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers | journal = Molecular Microbiology | volume = 79 | issue = 1 | pages = 35–49 | date = January 2011 | pmid = 21166892 | pmc = 3025118 | doi = 10.1111/j.1365-2958.2010.07452.x }}</ref><ref name="pmid21385233">{{cite journal | vauthors = Manica A, Zebec Z, Teichmann D, Schleper C | title = In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon | journal = Molecular Microbiology | volume = 80 | issue = 2 | pages = 481–491 | date = April 2011 | pmid = 21385233 | doi = 10.1111/j.1365-2958.2011.07586.x | doi-access = free }}</ref> The type I-E complex (commonly referred to as Cascade) requires five Cas proteins bound to a single crRNA.<ref name="pmid21460843">{{cite journal | vauthors = Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul U, Wurm R, Wagner R, Beijer MR, Barendregt A, Zhou K, Snijders AP, Dickman MJ, Doudna JA, Boekema EJ, Heck AJ, van der Oost J, Brouns SJ | author-link16 = Jennifer Doudna | title = Structural basis for CRISPR RNA-guided DNA recognition by Cascade | journal = Nature Structural & Molecular Biology | volume = 18 | issue = 5 | pages = 529–536 | date = May 2011 | pmid = 21460843 | doi = 10.1038/nsmb.2019 | url = https://pure.rug.nl/ws/files/6761944/2011NatStructMolBiolJore.pdf }}</ref><ref name="pmid21938068">{{cite journal | vauthors = Wiedenheft B, Lander GC, Zhou K, Jore MM, Brouns SJ, van der Oost J, Doudna JA, Nogales E | author-link7=Jennifer Doudna | title = Structures of the RNA-guided surveillance complex from a bacterial immune system | journal = Nature | volume = 477 | issue = 7365 | pages = 486–489 | date = September 2011 | pmid = 21938068 | doi = 10.1038/nature10402 | bibcode = 2011Natur.477..486W | pmc=4165517}}</ref>

=== Interference ===
During the interference stage in type I systems, the PAM sequence is recognized on the crRNA-complementary strand and is required along with crRNA annealing. In type I systems correct base pairing between the crRNA and the protospacer signals a conformational change in Cascade that recruits [[Cas3]] for DNA degradation.

Type II systems rely on a single multifunctional protein, [[Cas9]], for the interference step.<ref name="pmid22949671"/> Cas9 requires both the crRNA and the tracrRNA to function and cleave DNA using its dual HNH and RuvC/RNaseH-like endonuclease domains. Basepairing between the PAM and the phage genome is required in type II systems. However, the PAM is recognized on the same strand as the crRNA (the opposite strand to type I systems).

Type III systems, like type I require six or seven Cas proteins binding to crRNAs.<ref name="pmid22227115">{{cite journal | vauthors = Zhang J, Rouillon C, Kerou M, Reeks J, Brugger K, Graham S, Reimann J, Cannone G, Liu H, Albers SV, Naismith JH, Spagnolo L, White MF | title = Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity | journal = Molecular Cell | volume = 45 | issue = 3 | pages = 303–313 | date = February 2012 | pmid = 22227115 | pmc = 3381847 | doi = 10.1016/j.molcel.2011.12.013 }}</ref><ref name="pmid19945378">{{cite journal | vauthors = Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP | title = RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex | journal = Cell | volume = 139 | issue = 5 | pages = 945–956 | date = November 2009 | pmid = 19945378 | pmc = 2951265 | doi = 10.1016/j.cell.2009.07.040 }}</ref> The type III systems analysed from ''S. solfataricus'' and ''P. furiosus'' both target the mRNA of phages rather than phage DNA genome,<ref name="pmid23320564"/><ref name="pmid19945378"/> which may make these systems uniquely capable of targeting RNA-based phage genomes.<ref name="pmid22337052"/> Type III systems were also found to target DNA in addition to RNA using a different Cas protein in the complex, Cas10.<ref name="Estrella 2016">{{Cite journal |vauthors=Estrella MA, Kuo FT, Bailey S| doi = 10.1101/gad.273722.115| title =RNA-activated DNA cleavage by the Type III-B CRISPR–Cas effector complex| journal = [[Genes & Development]] | volume = 30 | issue = 4 | pages = 460–470 | year = 2016 | pmid = 26848046| pmc = 4762430 }}</ref> The DNA cleavage was shown to be transcription dependent.<ref name="Samai 2015">{{Cite journal |vauthors=Samai P, Pyenson N, Jiang W, Goldberg GW, Hatoum-Aslan A, Marraffini LA|author2-link=Nicholas Pyenson| doi = 10.1016/j.cell.2015.04.027| title =Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity| journal = Cell | volume = 161 | issue = 5 | pages = 1164–1174 | year = 2015 | pmid =25959775| pmc = 4594840 }}</ref>

The mechanism for distinguishing self from foreign DNA during interference is built into the crRNAs and is therefore likely common to all three systems. Throughout the distinctive maturation process of each major type, all crRNAs contain a spacer sequence and some portion of the repeat at one or both ends. It is the partial repeat sequence that prevents the CRISPR-Cas system from targeting the chromosome as base pairing beyond the spacer sequence signals self and prevents DNA cleavage.<ref name="pmid20072129">{{cite journal | vauthors = Marraffini LA, Sontheimer EJ | title = Self versus non-self discrimination during CRISPR RNA-directed immunity | journal = Nature | volume = 463 | issue = 7280 | pages = 568–571 | date = January 2010 | pmid = 20072129 | pmc = 2813891 | doi = 10.1038/nature08703 | bibcode = 2010Natur.463..568M }}</ref> RNA-guided CRISPR enzymes are classified as [[Restriction enzyme#Type V|type V restriction enzymes]].

== Evolution ==
{{Infobox protein family
| Symbol = CRISPR_Cas2
| Name = CRISPR associated protein Cas2 (adaptation RNase)
| image = PDB 1zpw EBI.jpg
| width =
| caption = Crystal structure of a hypothetical protein tt1823 from Thermus thermophilus
| Pfam = PF09827
| Pfam_clan =
| InterPro = IPR019199
| SMART =
| PROSITE =
| MEROPS =
| SCOP =
| TCDB =
| OPM family =
| OPM protein =
| CAZy =
| CDD = cd09638
}}

{{Infobox protein family
| Symbol = CRISPR_Cse1
| Name = CRISPR-associated protein CasA/Cse1 (Type I effector DNase)
| image =
| width =
| caption =
| Pfam = PF09481
| Pfam_clan =
| InterPro = IPR013381
| SMART =
| PROSITE =
| MEROPS =
| SCOP =
| TCDB =
| OPM family =
| OPM protein =
| CAZy =
| CDD = cd09729
}}
{{Infobox protein family
| Symbol = CRISPR_assoc
| Name = CRISPR associated protein CasC/Cse3/Cas6 (Type I effector RNase)
| image = PDB 1wj9 EBI.jpg
| width =
| caption = Crystal structure of a crispr-associated protein from Thermus thermophilus
| Pfam = PF08798
| Pfam_clan = CL0362
| InterPro = IPR010179
| SMART =
| PROSITE =
| MEROPS =
| SCOP =
| TCDB =
| OPM family =
| OPM protein =
| CAZy =
| CDD = cd09727
}}

The cas genes in the adaptor and effector modules of the CRISPR-Cas system are believed to have evolved from two different ancestral modules. A [[transposon]]-like element called [[casposon]] encoding the Cas1-like integrase and potentially other components of the adaptation module was inserted next to the ancestral effector module, which likely functioned as an independent innate immune system.<ref>{{cite journal | vauthors = Krupovic M, Béguin P, Koonin EV | author-link3=Eugene Koonin | title = Casposons: mobile genetic elements that gave rise to the CRISPR-Cas adaptation machinery | journal = Current Opinion in Microbiology | volume = 38 | pages = 36–43 | date = August 2017 | pmid = 28472712 | pmc = 5665730 | doi = 10.1016/j.mib.2017.04.004 }}</ref> The highly conserved cas1 and cas2 genes of the adaptor module evolved from the ancestral module while a variety of class 1 effector cas genes evolved from the ancestral effector module.<ref>{{cite journal | vauthors = Koonin EV, Makarova KS | author-link1=Eugene Koonin | title = CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes | journal = RNA Biology | volume = 10 | issue = 5 | pages = 679–686 | date = May 2013 | pmid = 23439366 | pmc = 3737325 | doi = 10.4161/rna.24022 }}</ref> The evolution of these various class 1 effector module cas genes was guided by various mechanisms, such as duplication events.<ref>{{cite journal | vauthors = Koonin EV, Makarova KS, Zhang F | author-link=Eugene Koonin | author-link3=Feng Zhang | title = Diversity, classification and evolution of CRISPR-Cas systems | journal = Current Opinion in Microbiology | volume = 37 | pages = 67–78 | date = June 2017 | pmid = 28605718 | pmc = 5776717 | doi = 10.1016/j.mib.2017.05.008 }}</ref> On the other hand, each type of class 2 effector module arose from subsequent independent insertions of mobile genetic elements.<ref name="Shmakov-2017">{{cite journal | vauthors = Shmakov S, Smargon A, Scott D, Cox D, Pyzocha N, Yan W, Abudayyeh OO, Gootenberg JS, Makarova KS, Wolf YI, Severinov K, Zhang F, Koonin EV | author-link13=Eugene Koonin | author-link12=Feng Zhang | title = Diversity and evolution of class 2 CRISPR-Cas systems | journal = Nature Reviews. Microbiology | volume = 15 | issue = 3 | pages = 169–182 | date = March 2017 | pmid = 28111461 | pmc = 5851899 | doi = 10.1038/nrmicro.2016.184 }}</ref> These mobile genetic elements took the place of the multiple gene effector modules to create single gene effector modules that produce large proteins which perform all the necessary tasks of the effector module.<ref name="Shmakov-2017"/> The spacer regions of CRISPR-Cas systems are taken directly from foreign mobile genetic elements and thus their long-term evolution is hard to trace.<ref>{{cite journal | vauthors = Kupczok A, Bollback JP | title = Probabilistic models for CRISPR spacer content evolution | journal = BMC Evolutionary Biology | volume = 13 | issue = 1 | page = 54 | date = February 2013 | pmid = 23442002 | pmc = 3704272 | doi = 10.1186/1471-2148-13-54 | bibcode = 2013BMCEE..13...54K | doi-access = free }}</ref> The non-random evolution of these spacer regions has been found to be highly dependent on the environment and the particular foreign mobile genetic elements it contains.<ref>{{cite journal | vauthors = Sternberg SH, Richter H, Charpentier E, Qimron U | title = Adaptation in CRISPR-Cas Systems | journal = Molecular Cell | volume = 61 | issue = 6 | pages = 797–808 | date = March 2016 | pmid = 26949040 | doi = 10.1016/j.molcel.2016.01.030 | hdl = 21.11116/0000-0003-E74E-2 | hdl-access = free }}</ref>

CRISPR-Cas can immunize bacteria against certain phages and thus halt transmission. For this reason, [[Eugene Koonin|Koonin]] described CRISPR-Cas as a [[Lamarckism|Lamarckian]] inheritance mechanism.<ref name="pmid19906303">{{cite journal | vauthors = Koonin EV, Wolf YI | author-link=Eugene Koonin | title = Is evolution Darwinian or/and Lamarckian? | journal = Biology Direct | volume = 4 | page = 42 | date = November 2009 | pmid = 19906303 | pmc = 2781790 | doi = 10.1186/1745-6150-4-42 | doi-access=free }}</ref> However, this was disputed by a critic who noted, "We should remember [Lamarck] for the good he contributed to science, not for things that resemble his theory only superficially. Indeed, thinking of CRISPR and other phenomena as Lamarckian only obscures the simple and elegant way evolution really works".<ref>{{cite journal | vauthors = Weiss A | title = Lamarckian Illusions | journal = Trends in Ecology & Evolution | volume = 30 | issue = 10 | pages = 566–568 | date = October 2015 | pmid = 26411613 | doi = 10.1016/j.tree.2015.08.003 | doi-access = free | bibcode = 2015TEcoE..30..566W }}</ref> But as more recent studies have been conducted, it has become apparent that the acquired spacer regions of CRISPR-Cas systems are indeed a form of Lamarckian evolution because they are genetic mutations that are acquired and then passed on.<ref name="Koonin-2016">{{cite journal | vauthors = Koonin EV, Wolf YI | author-link=Eugene Koonin | title = Just how Lamarckian is CRISPR-Cas immunity: the continuum of evolvability mechanisms | journal = Biology Direct | volume = 11 | issue = 1 | page = 9 | date = February 2016 | pmid = 26912144 | pmc = 4765028 | doi = 10.1186/s13062-016-0111-z | doi-access=free }}</ref> On the other hand, the evolution of the Cas gene machinery that facilitates the system evolves through classic Darwinian evolution.<ref name="Koonin-2016"/>

=== Coevolution ===
Analysis of CRISPR sequences revealed [[coevolution]] of host and viral genomes.<ref>{{cite journal | vauthors = Heidelberg JF, Nelson WC, Schoenfeld T, Bhaya D | title = Germ warfare in a microbial mat community: CRISPRs provide insights into the co-evolution of host and viral genomes | journal = PLOS ONE | volume = 4 | issue = 1 | pages = e4169 | year = 2009 | pmid = 19132092 | pmc = 2612747 | doi = 10.1371/journal.pone.0004169 | bibcode = 2009PLoSO...4.4169H | veditors = Ahmed N | doi-access = free }}</ref>

The basic model of CRISPR evolution is newly incorporated spacers driving phages to mutate their genomes to avoid the bacterial immune response, creating diversity in both the phage and host populations. To resist a phage infection, the sequence of the CRISPR spacer must correspond perfectly to the sequence of the target phage gene. Phages can continue to infect their hosts' given point mutations in the spacer.<ref name="pmid20072129"/> Similar stringency is required in PAM or the bacterial strain remains phage sensitive.<ref name="pmid18065545"/><ref name="pmid20072129"/>

=== Rates ===
A study of 124 ''S. thermophilus'' strains showed that 26% of all spacers were unique and that different CRISPR loci showed different rates of spacer acquisition.<ref name="pmid18065539"/> Some CRISPR loci evolve more rapidly than others, which allowed the strains' phylogenetic relationships to be determined. A [[Comparative genomics|comparative genomic]] analysis showed that ''E. coli'' and ''[[Salmonella enterica|S. enterica]]'' evolve much more slowly than ''S. thermophilus''. The latter's strains that diverged 250,000 years ago still contained the same spacer complement.<ref name="pmid20559554">{{cite journal | vauthors = Touchon M, Rocha EP | title = The small, slow and specialized CRISPR and anti-CRISPR of Escherichia and Salmonella | journal = PLOS ONE | volume = 5 | issue = 6 | pages = e11126 | date = June 2010 | pmid = 20559554 | pmc = 2886076 | doi = 10.1371/journal.pone.0011126 | veditors = Randau L | bibcode = 2010PLoSO...511126T | doi-access = free }}</ref>

[[metagenomics|Metagenomic]] analysis of two acid-mine-drainage [[biofilm]]s showed that one of the analyzed CRISPRs contained extensive deletions and spacer additions versus the other biofilm, suggesting a higher phage activity/prevalence in one community than the other.<ref name="pmid17894817"/> In the oral cavity, a temporal study determined that 7–22% of spacers were shared over 17 months within an individual while less than 2% were shared across individuals.<ref name="pmid21149389"/>

From the same environment, a single strain was tracked using [[Polymerase chain reaction|PCR]] primers specific to its CRISPR system. Broad-level results of spacer presence/absence showed significant diversity. However, this CRISPR added three spacers over 17 months,<ref name="pmid21149389"/> suggesting that even in an environment with significant CRISPR diversity some loci evolve slowly.

CRISPRs were analysed from the metagenomes produced for the [[Human Microbiome Project]].<ref name="pmid22719260">{{cite journal | vauthors = Rho M, Wu YW, Tang H, Doak TG, Ye Y | title = Diverse CRISPRs evolving in human microbiomes | journal = PLOS Genetics | volume = 8 | issue = 6 | pages = e1002441 | year = 2012 | pmid = 22719260 | pmc = 3374615 | doi = 10.1371/journal.pgen.1002441 | doi-access = free }}</ref> Although most were body-site specific, some within a body site are widely shared among individuals. One of these loci originated from [[streptococcal]] species and contained ≈15,000 spacers, 50% of which were unique. Similar to the targeted studies of the oral cavity, some showed little evolution over time.<ref name="pmid22719260"/>

CRISPR evolution was studied in [[chemostat]]s using ''S. thermophilus'' to directly examine spacer acquisition rates. In one week, ''S. thermophilus'' strains acquired up to three spacers when challenged with a single phage.<ref name="pmid23057534">{{cite journal | vauthors = Sun CL, Barrangou R, Thomas BC, Horvath P, Fremaux C, Banfield JF | author-link2=Rodolphe Barrangou | title = Phage mutations in response to CRISPR diversification in a bacterial population | journal = Environmental Microbiology | volume = 15 | issue = 2 | pages = 463–470 | date = February 2013 | pmid = 23057534 | doi = 10.1111/j.1462-2920.2012.02879.x | bibcode=2013EnvMi..15..463S }}</ref> During the same interval, the phage developed [[single-nucleotide polymorphism]]s that became fixed in the population, suggesting that targeting had prevented phage replication absent these mutations.<ref name="pmid23057534"/>

Another ''S. thermophilus'' experiment showed that phages can infect and replicate in hosts that have only one targeting spacer. Yet another showed that sensitive hosts can exist in environments with high-phage titres.<ref name="pmid24586036">{{cite journal | vauthors = Kuno S, Sako Y, Yoshida T | title = Diversification of CRISPR within coexisting genotypes in a natural population of the bloom-forming cyanobacterium Microcystis aeruginosa | journal = Microbiology | volume = 160 | issue = Pt 5 | pages = 903–916 | date = May 2014 | pmid = 24586036 | doi = 10.1099/mic.0.073494-0 | doi-access = free }}</ref> The chemostat and observational studies suggest many nuances to CRISPR and phage (co)evolution.

== Identification ==
CRISPRs are widely distributed among bacteria and archaea<ref name="pmid24728998"/> and show some sequence similarities.<ref name="pmid17442114"/> Their most notable characteristic is their repeating spacers and direct repeats. This characteristic makes CRISPRs easily identifiable in long sequences of DNA, since the number of repeats decreases the likelihood of a false positive match.<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 = 181–186 | date = March 2008 | pmid = 18157154 | doi = 10.1038/nrmicro1793 | url = https://digital.library.unt.edu/ark:/67531/metadc893045/ | quote = Table 1: Web resources for CRISPR analysis }}</ref>

Analysis of CRISPRs in metagenomic data is more challenging, as CRISPR loci do not typically assemble, due to their repetitive nature or through strain variation, which confuses assembly algorithms. Where many reference genomes are available, [[polymerase chain reaction]] (PCR) can be used to amplify CRISPR arrays and analyse spacer content.<ref name="pmid18065539"/><ref name="pmid21149389"/><ref name="pmid22583485">{{cite journal | vauthors = Pride DT, Salzman J, Relman DA | title = Comparisons of clustered regularly interspaced short palindromic repeats and viromes in human saliva reveal bacterial adaptations to salivary viruses | journal = Environmental Microbiology | volume = 14 | issue = 9 | pages = 2564–2576 | date = September 2012 | pmid = 22583485 | pmc = 3424356 | doi = 10.1111/j.1462-2920.2012.02775.x | bibcode = 2012EnvMi..14.2564P }}</ref><ref name="pmid23701169">{{cite journal | vauthors = Held NL, Herrera A, Whitaker RJ | title = Reassortment of CRISPR repeat-spacer loci in Sulfolobus islandicus | journal = Environmental Microbiology | volume = 15 | issue = 11 | pages = 3065–3076 | date = November 2013 | pmid = 23701169 | doi = 10.1111/1462-2920.12146 | bibcode = 2013EnvMi..15.3065H }}</ref><ref name="pmid20927396">{{cite journal | vauthors = Held NL, Herrera A, Cadillo-Quiroz H, Whitaker RJ | title = CRISPR associated diversity within a population of Sulfolobus islandicus | journal = PLOS ONE | volume = 5 | issue = 9 | pages = e12988 | date = September 2010 | pmid = 20927396 | pmc = 2946923 | doi = 10.1371/journal.pone.0012988 | bibcode = 2010PLoSO...512988H | doi-access = free }}</ref><ref name="pmid31729390">{{cite journal | vauthors = Medvedeva S, Liu Y, Koonin EV, Severinov K, Prangishvili D, Krupovic M | title = Virus-borne mini-CRISPR arrays are involved in interviral conflicts | journal = Nature Communications | volume = 10 | issue = 1 | page = 5204 | date = November 2019 | pmid = 31729390 | pmc = 6858448 | doi = 10.1038/s41467-019-13205-2 | bibcode = 2019NatCo..10.5204M }}</ref> However, this approach yields information only for specifically targeted CRISPRs and for organisms with sufficient representation in public databases to design reliable polymerase PCR primers. Degenerate repeat-specific primers can be used to amplify CRISPR spacers directly from environmental samples; amplicons containing two or three spacers can be then computationally assembled to reconstruct long CRISPR arrays.<ref name="pmid31729390"/>

The alternative is to extract and reconstruct CRISPR arrays from shotgun metagenomic data. This is computationally more difficult, particularly with second generation sequencing technologies (e.g. 454, Illumina), as the short read lengths prevent more than two or three repeat units appearing in a single read. CRISPR identification in raw reads has been achieved using purely ''de novo'' identification<ref name="pmid23511966">{{cite journal | vauthors = Skennerton CT, Imelfort M, Tyson GW | title = Crass: identification and reconstruction of CRISPR from unassembled metagenomic data | journal = Nucleic Acids Research | volume = 41 | issue = 10 | pages = e105 | date = May 2013 | pmid = 23511966 | pmc = 3664793 | doi = 10.1093/nar/gkt183 }}</ref> or by using direct repeat sequences in partially assembled CRISPR arrays from [[contig]]s (overlapping DNA segments that together represent a consensus region of DNA)<ref name="pmid22719260"/> and direct repeat sequences from published genomes<ref name="pmid22732228">{{cite journal | vauthors = Stern A, Mick E, Tirosh I, Sagy O, Sorek R | title = CRISPR targeting reveals a reservoir of common phages associated with the human gut microbiome | journal = Genome Research | volume = 22 | issue = 10 | pages = 1985–1994 | date = October 2012 | pmid = 22732228 | pmc = 3460193 | doi = 10.1101/gr.138297.112 }}</ref> as a hook for identifying direct repeats in individual reads.

== Use by phages ==
Another way for bacteria to defend against phage infection is by having [[genomic island|chromosomal islands]]. A subtype of chromosomal islands called phage-inducible chromosomal island (PICI) is excised from a bacterial chromosome upon phage infection and can inhibit phage replication.<ref name="pmid20634809">{{cite journal | vauthors = Novick RP, Christie GE, Penadés JR | title = The phage-related chromosomal islands of Gram-positive bacteria | journal = Nature Reviews Microbiology | volume = 8 | issue = 8 | pages = 541–551 | date = August 2010 | pmid = 20634809 | pmc = 3522866 | doi = 10.1038/nrmicro2393 }}</ref> PICIs are induced, excised, replicated, and finally packaged into small capsids by certain staphylococcal temperate phages. PICIs use several mechanisms to block phage reproduction. In the first mechanism, PICI-encoded Ppi differentially blocks phage maturation by binding or interacting specifically with phage TerS, hence blocking phage TerS/TerL complex formation responsible for phage DNA packaging. In the second mechanism PICI CpmAB redirects the phage capsid morphogenetic protein to make 95% of SaPI-sized capsid and phage DNA can package only 1/3rd of their genome in these small capsids and hence become nonviable phage.<ref>{{cite journal|vauthors=Ram G, Chen J, Kumar K, Ross HF, Ubeda C, Damle PK, Lane KD, Penadés JR, Christie GE, Novick RP|date=October 2012|title=Staphylococcal pathogenicity island interference with helper phage reproduction is a paradigm of molecular parasitism|journal=Proceedings of the National Academy of Sciences of the United States of America|volume=109|issue=40|pages=16300–16305|doi=10.1073/pnas.1204615109|pmc=3479557|pmid=22991467|bibcode=2012PNAS..10916300R|doi-access=free}}</ref> The third mechanism involves two proteins, PtiA and PtiB, that target the LtrC, which is responsible for the production of virion and lysis proteins. This interference mechanism is modulated by a modulatory protein, PtiM, binds to one of the interference-mediating proteins, PtiA, and hence achieves the required level of interference.<ref>{{cite journal|vauthors=Ram G, Chen J, Ross HF, Novick RP|date=October 2014|title=Precisely modulated pathogenicity island interference with late phage gene transcription|journal=Proceedings of the National Academy of Sciences of the United States of America|volume=111|issue=40|pages=14536–14541|doi=10.1073/pnas.1406749111|pmc=4209980|pmid=25246539|bibcode=2014PNAS..11114536R|doi-access=free}}</ref>

One study showed that lytic ICP1 phage, which specifically targets ''[[Vibrio cholerae]]'' [[serogroup]] O1, has acquired a CRISPR-Cas system that targets a ''V. cholera'' PICI-like element. The system has 2 CRISPR loci and 9 Cas genes. It seems to be [[Homology (biology)|homologous]] to the I-F system found in ''[[Yersinia pestis]]''. Moreover, like the bacterial CRISPR-Cas system, ICP1 CRISPR-Cas can acquire new sequences, which allows phage and host to co-evolve.<ref name="pmid23446421">{{cite journal | vauthors = Seed KD, Lazinski DW, Calderwood SB, Camilli A | title = A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity | journal = Nature | volume = 494 | issue = 7438 | pages = 489–491 | date = February 2013 | pmid = 23446421 | pmc = 3587790 | doi = 10.1038/nature11927 | bibcode = 2013Natur.494..489S }}</ref><ref name="Boyd">{{cite journal | vauthors = Boyd CM, Angermeyer A, Hays SG, Barth ZK, Patel KM, Seed KD | title = Bacteriophage ICP1: A Persistent Predator of ''Vibrio cholerae'' | journal = Annual Review of Virology | volume = 8 | issue = 1 | pages = 285–304 | date = September 2021 | pmid = 34314595 | doi = 10.1146/annurev-virology-091919-072020 | pmc = 9040626 |issn=2327-056X | doi-access = free }}</ref>

Certain archaeal viruses were shown to carry mini-CRISPR arrays containing one or two spacers. It has been shown that spacers within the virus-borne CRISPR arrays target other viruses and plasmids, suggesting that mini-CRISPR arrays represent a mechanism of heterotypic superinfection exclusion and participate in interviral conflicts.<ref name="pmid31729390"/>

== Applications ==
{{main|CRISPR gene editing}}

{{cs1 config|name-list-style=vanc|display-authors=6}}
CRISPR gene editing is a revolutionary technology that allows for precise, targeted modifications to the DNA of living organisms. Developed from a natural defense mechanism found in bacteria, CRISPR-Cas9 is the most commonly used system. Gene editing with CRISPR-Cas9 involves a [[Cas9|Cas9 nuclease]] and an engineered [[guide RNA]], which come together to allow for the precise "cutting" of one or both strands of DNA at specific locations within the genome.<ref name="Koblan_2020">{{Cite journal | vauthors = Koblan LW, Liu DR, Anzalone AV | title = Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors | journal = Nature Biotechnology | volume = 38 | issue = 7 | pages = 824–844 | date = July 2020 | pmid = 32572269 | doi = 10.1038/s41587-020-0561-9 | url = https://www.nature.com/articles/s41587-020-0561-9 | language = en | issn = 1546-1696 }}</ref> It makes use of the cell's natural DNA repair systems, including [[non-homologous end joining]], [[Homology directed repair|homology-directed repair]], or [[DNA mismatch repair|mismatch repair]], to modify, insert, or delete genetic material at these specific cut sites. <ref name="Koblan_2020" /><ref>{{Cite journal | vauthors = Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR, Anzalone AV | title = Search-and-replace genome editing without double-strand breaks or donor DNA | journal = Nature | volume = 576 | issue = 7785 | pages = 149–157 | date = December 2019 | pmid = 31634902 | pmc = 6907074 | doi = 10.1038/s41586-019-1711-4 | url = https://www.nature.com/articles/s41586-019-1711-4 | language = en | issn = 1476-4687 }}</ref> This technology has transformed fields such as genetics, medicine,<ref name="Feng_2024">{{cite journal | vauthors = Feng Q, Li Q, Zhou H, Wang Z, Lin C, Jiang Z, Liu T, Wang D | title = CRISPR technology in human diseases | journal = MedComm | volume = 5 | issue = 8 | pages = e672 | date = August 2024 | pmid = 39081515 | pmc = 11286548 | doi = 10.1002/mco2.672 }}</ref><ref name="Li_2024">{{cite journal | vauthors = Li T, Li S, Kang Y, Zhou J, Yi M | title = Harnessing the evolving CRISPR/Cas9 for precision oncology | journal = Journal of Translational Medicine | volume = 22 | issue = 1 | pages = 749 | date = August 2024 | pmid = 39118151 | pmc = 11312220 | doi = 10.1186/s12967-024-05570-4 | doi-access = free }}</ref><ref name="Kaur_2024">{{cite journal | vauthors = Kaur R, Gupta S, Chauhan A, Mishra V, Sharma MK, Singh J | title = Harnessing the power of clustered regularly interspaced short palindromic repeats (CRISPR) based microfluidics for next-generation molecular diagnostics | journal = Molecular Biology Reports | volume = 51 | issue = 1 | pages = 896 | date = August 2024 | pmid = 39115550 | doi = 10.1007/s11033-024-09840-8 }}</ref> and agriculture,<ref name="Mishra_2024">{{cite journal | vauthors = Mishra S, Nayak S, Tuteja N, Poosapati S, Swain DM, Sahoo RK | title = CRISPR/Cas-Mediated Genome Engineering in Plants: Application and Prospectives | journal = Plants | location = Basel, Switzerland | volume = 13 | issue = 14 | pages = 1884 | date = July 2024 | pmid = 39065411 | pmc = 11279650 | doi = 10.3390/plants13141884 | doi-access = free | bibcode = 2024Plnts..13.1884M | publication-place = Basel, Switzerland }}</ref> offering potential treatments for genetic disorders, advancements in crop engineering, and research into the fundamental workings of life. However, its ethical implications and potential unintended consequences have sparked significant debate.<ref name="Shinwari_2018">{{cite journal | vauthors = Shinwari ZK, Tanveer F, Khalil AT | title = Ethical Issues Regarding CRISPR Mediated Genome Editing | journal = Current Issues in Molecular Biology | volume = 26 | pages = 103–110 | date = 2018 | pmid = 28879860 | doi = 10.21775/cimb.026.103 | doi-access = free }}</ref><ref name="Kozan_2024">{{cite journal | vauthors = Kozan DW, Farber SA | title = Is It Ever Wise to Edit Wild-Type Alleles? Engineered CRISPR Alleles Versus Millions of Years of Human Evolution | journal = Arteriosclerosis, Thrombosis, and Vascular Biology | volume = 44 | issue = 2 | pages = 328–333 | date = February 2024 | pmid = 38059350 | pmc = 10948015 | doi = 10.1161/ATVBAHA.123.318069 }}</ref>

== See also ==
{{div col|colwidth=18em}}

* [[CRISPR activation]]
* [[Anti-CRISPR]]
* [[CRISPR/Cas Tools]]
* [[CRISPR gene editing]]
* [[The CRISPR Journal]]
* "[[Designer baby]]"
* [[DRACO]]
* [[Gene knockout]]
* [[Genome-wide CRISPR-Cas9 knockout screens]]
* [[Glossary of genetics]]
* [[Human germline engineering]]
* [[Human Nature (2019 film)|''Human Nature'' (2019 documentary film)]]
* [[MAGESTIC]]
* [[New eugenics|''New'' eugenics]]
* [[Prime editing]]
* [[RNAi]]
* [[SiRNA]]
* [[Surveyor nuclease assay]]
* [[Synthetic biology]]
* [[Zinc finger]]
{{div col end}}

==Notes==
{{Reflist|group=Note}}

{{clear}}

== References ==
{{reflist}}

== Further reading ==
{{Refbegin|30em}}
* {{cite book | vauthors = Doudna J, Mali P | author-link1 = Jennifer Doudna | title = CRISPR-Cas: A Laboratory Manual | date = 23 March 2016 | publisher = Cold Spring Harbor Laboratory Press | location = New York | isbn = 978-1-62182-131-1 }}
* {{cite journal | vauthors = Mohanraju P, Makarova KS, Zetsche B, Zhang F, Koonin EV, van der Oost J | author-link4 = Feng Zhang | author-link5 = Eugene Koonin | title = Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems | journal = Science | volume = 353 | issue = 6299 | pages = aad5147 | date = August 2016 | pmid = 27493190 | doi = 10.1126/science.aad5147 | hdl = 1721.1/113195 | hdl-access = free }}
* {{cite journal | vauthors = Sander JD, Joung JK | title = CRISPR-Cas systems for editing, regulating and targeting genomes | journal = Nature Biotechnology | volume = 32 | issue = 4 | pages = 347–355 | date = April 2014 | pmid = 24584096 | pmc = 4022601 | doi = 10.1038/nbt.2842 }}
* {{cite journal | vauthors = Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F | author-link6=Feng Zhang | title = Rationally engineered Cas9 nucleases with improved specificity | journal = Science | volume = 351 | issue = 6268 | pages = 84–88 | date = January 2016 | pmid =  26628643| doi = 10.1126/science.aad5227 | pmc=4714946| bibcode = 2016Sci...351...84S }}
* {{cite journal | vauthors = Terns RM, Terns MP | title = CRISPR-based technologies: prokaryotic defense weapons repurposed | journal = Trends in Genetics | volume = 30 | issue = 3 | pages = 111–118 | date = March 2014 | pmid = 24555991 | pmc = 3981743 | doi = 10.1016/j.tig.2014.01.003 }}
* {{cite journal | vauthors = Westra ER, Buckling A, Fineran PC | title = CRISPR-Cas systems: beyond adaptive immunity | journal = Nature Reviews Microbiology | volume = 12 | issue = 5 | pages = 317–326 | date = May 2014 | pmid = 24704746 | doi = 10.1038/nrmicro3241 }}
* {{cite journal | vauthors = Andersson AF, Banfield JF | title = Virus population dynamics and acquired virus resistance in natural microbial communities | journal = Science | volume = 320 | issue = 5879 | pages = 1047–1050 | date = May 2008 | pmid = 18497291 | doi = 10.1126/science.1157358 | bibcode = 2008Sci...320.1047A }}
* {{cite journal | vauthors = Hale C, Kleppe K, Terns RM, Terns MP | title = Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus | journal = RNA | volume = 14 | issue = 12 | pages = 2572–2579 | date = December 2008 | pmid = 18971321 | pmc = 2590957 | doi = 10.1261/rna.1246808 }}
* {{cite journal | vauthors = van der Ploeg JR | title = Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages | journal = Microbiology | volume = 155 | issue = Pt 6 | pages = 1966–1976 | date = June 2009 | pmid = 19383692 | doi = 10.1099/mic.0.027508-0 | doi-access = free }}
* {{cite journal | vauthors = van der Oost J, Brouns SJ | title = RNAi: prokaryotes get in on the act | journal = Cell | volume = 139 | issue = 5 | pages = 863–865 | date = November 2009 | pmid = 19945373 | doi = 10.1016/j.cell.2009.11.018 | doi-access = free }}
* {{cite journal | vauthors = Karginov FV, Hannon GJ | title = The CRISPR system: small RNA-guided defense in bacteria and archaea | journal = Molecular Cell | volume = 37 | issue = 1 | pages = 7–19 | date = January 2010 | pmid = 20129051 | pmc = 2819186 | doi = 10.1016/j.molcel.2009.12.033 }}
* {{cite journal | vauthors = Pul U, Wurm R, Arslan Z, Geissen R, Hofmann N, Wagner R | title = Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS | journal = Molecular Microbiology | volume = 75 | issue = 6 | pages = 1495–1512 | date = March 2010 | pmid = 20132443 | doi = 10.1111/j.1365-2958.2010.07073.x | doi-access = free }}
* {{cite journal | vauthors = Díez-Villaseñor C, Almendros C, García-Martínez J, Mojica FJ | title = Diversity of CRISPR loci in ''Escherichia coli'' | journal = Microbiology | volume = 156 | issue = Pt 5 | pages = 1351–1361 | date = May 2010 | pmid = 20133361 | doi = 10.1099/mic.0.036046-0 | doi-access = free }}
* {{cite journal | vauthors = Deveau H, Garneau JE, Moineau S | title = CRISPR/Cas system and its role in phage-bacteria interactions | journal = Annual Review of Microbiology | volume = 64 | pages = 475–493 | year = 2010 | pmid = 20528693 | doi = 10.1146/annurev.micro.112408.134123 }}
* {{cite journal | vauthors = Koonin EV, Makarova KS | author-link=Eugene Koonin | title = CRISPR-Cas: an adaptive immunity system in prokaryotes | journal = F1000 Biology Reports | volume = 1 | page = 95 | date = December 2009 | pmid = 20556198 | pmc = 2884157 | doi = 10.3410/B1-95 | doi-access=free }}
* {{Cite news|title = The age of the red pen|url = https://www.economist.com/news/briefing/21661799-it-now-easy-edit-genomes-plants-animals-and-humans-age-red-pen|newspaper = The Economist|access-date = 2015-08-25|issn = 0013-0613|date = August 22, 2015|archive-date = 2015-08-24|archive-url = https://web.archive.org/web/20150824054220/http://www.economist.com/news/briefing/21661799-it-now-easy-edit-genomes-plants-animals-and-humans-age-red-pen|url-status = live}}
* {{cite journal | vauthors = Ran AF, 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 = 2281–2308 | date = 2013 | pmid = 24157548 | pmc = 3969860 | doi = 10.1038/nprot.2013.143 }}
{{refend}}

== External links ==
{{Commons category}}
{{Scholia|topic}}
* {{cite web|url=https://fas.org/sgp/crs/misc/R44824.pdf |title=Advanced Gene Editing: CRISPR-Cas9 |publisher=[[Congressional Research Service]]}}
* {{cite web|url=https://www.ibiology.org/ibiomagazine/jennifer-doudna-genome-engineering-with-crispr-cas9-birth-of-a-breakthrough-technology.html |title=Jennifer Doudna talk: Genome Engineering with CRISPR-Cas9: Birth of a Breakthrough Technology|date=10 September 2022 }}
*{{cite episode|title=Human Nature|series=NOVA|series-link=Nova (American TV program)|network=[[PBS]]|station=[[WGBH-TV|WGBH]]|date=September 9, 2020|season=47|number=9|url=https://www.pbs.org/wgbh/nova/video/human-nature/|access-date=April 7, 2023}}

===Protein Data Bank===
* {{PDBe-KB2|Q46901|CRISPR system Cascade subunit CasA}}
* {{PDBe-KB2|P76632|CRISPR system Cascade subunit CasB}}
* {{PDBe-KB2|Q46899|CRISPR system Cascade subunit CasC}}
* {{PDBe-KB2|Q46898|CRISPR system Cascade subunit CasD}}
* {{PDBe-KB2|Q46897|CRISPR system Cascade subunit CasE}}

{{Glossaries of science and engineering}}
{{Repeated sequence}}
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[[Category:Biological engineering]]
[[Category:Biotechnology]]
[[Category:Genetic engineering]]
[[Category:Genome editing]]
[[Category:Jennifer Doudna]]
[[Category:Molecular biology]]
[[Category:Non-coding RNA]]
[[Category:Repetitive DNA sequences]]
[[Category:Immune system]]
[[Category:Prokaryote genes]]