CRISPR#Society and culture

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.{{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 }}{{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 }} The organization of the repeats was unusual. Repeated sequences are typically arranged consecutively, without interspersing different sequences. 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 repeats (DR) in that bacterium. They recognized the diversity of the sequences that intervened in the direct repeats among different strains of M. tuberculosis{{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 }} and used this property to design a typing method called spoligotyping, still in use today.{{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 }}{{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 }}

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.{{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 }} 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.{{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}} Because those sequences were interspaced, Mojica initially called these sequences "short regularly spaced repeats" (SRSR).{{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 }} 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.{{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 }} 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.{{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 }}{{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 }}

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.{{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 = }} 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.{{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 }}

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 motifs, suggesting a role in the dynamic structure of the CRISPR loci.{{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 }} 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.{{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 }}{{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 }}

In addition, several CRISPRs with similar sequences can be present in a single genome, only one of which is associated with cas genes.{{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 }}]]

In 2005, three independent research groups showed that some CRISPR spacers are derived from phage DNA and extrachromosomal DNA such as plasmids.{{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 }}{{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 }}{{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}} 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.{{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 }} All three studies proposing this idea were initially rejected by high-profile journals, but eventually appeared in other journals.{{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 }}

The first publication 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.{{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 }}

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. 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.{{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 }} 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.{{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 }} 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 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. A 2010 study showed that CRISPR-Cas cuts strands of both phage and plasmid DNA in S. thermophilus.{{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 }}

{{main|Cas9}}

A simpler CRISPR system from Streptococcus pyogenes relies on the protein Cas9. The Cas9 endonuclease is a four-component system that includes two small molecules: crRNA and trans-activating CRISPR RNA (tracrRNA).{{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 }}{{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 }} In 2012, Jennifer Doudna and Emmanuelle Charpentier re-engineered the Cas9 endonuclease into a more manageable two-component system by fusing the two RNA molecules into a "single-guide RNA" that, when combined with Cas9, could find and cut the DNA target specified by the guide RNA.{{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 }} This contribution was so significant that it was recognized by the Nobel Prize in Chemistry in 2020. By manipulating the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target any DNA sequence for separation. Another collaboration comprising Virginijus Šikšnys, Gasiūnas, Barrangou, and Horvath showed that Cas9 from the S. thermophilus CRISPR system can also be reprogrammed to target a site of their choosing by changing the sequence of its crRNA. These advances fueled efforts to edit genomes with the modified CRISPR-Cas9 system.

Groups led by Feng Zhang and George Church simultaneously published descriptions of genome editing in human cell cultures using CRISPR-Cas9 for the first time.{{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 }}{{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 }}{{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 }} It has since been used in a wide range of organisms, including baker's yeast (Saccharomyces cerevisiae),{{cite journal | vauthors = DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM | title = Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems | journal = Nucleic Acids Research | volume = 41 | issue = 7 | pages = 4336–4343 | date = April 2013 | pmid = 23460208 | pmc = 3627607 | doi = 10.1093/nar/gkt135 }}{{cite journal | vauthors = Zhang GC, Kong II, Kim H, Liu JJ, Cate JH, Jin YS | title = Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease | journal = Applied and Environmental Microbiology | volume = 80 | issue = 24 | pages = 7694–7701 | date = December 2014 | pmid = 25281382 | pmc = 4249234 | doi = 10.1128/AEM.02310-14 | bibcode = 2014ApEnM..80.7694Z }}{{cite journal | vauthors = Liu JJ, Kong II, Zhang GC, Jayakody LN, Kim H, Xia PF, Kwak S, Sung BH, Sohn JH, Walukiewicz HE, Rao CV, Jin YS | title = Metabolic Engineering of Probiotic Saccharomyces boulardii | journal = Applied and Environmental Microbiology | volume = 82 | issue = 8 | pages = 2280–2287 | date = April 2016 | pmid = 26850302 | pmc = 4959471 | doi = 10.1128/AEM.00057-16 | bibcode = 2016ApEnM..82.2280L }} the opportunistic pathogen Candida albicans,{{cite journal | vauthors = Vyas VK, Barrasa MI, Fink GR | title = Candida albicans CRISPR system permits genetic engineering of essential genes and gene families | journal = Science Advances | volume = 1 | issue = 3 | pages = e1500248 | date = 2015 | pmid = 25977940 | pmc = 4428347 | doi = 10.1126/sciadv.1500248 | bibcode = 2015SciA....1E0248V }}{{cite journal | vauthors = Ng H, Dean N | title = Candida albicans by Increased Single Guide RNA Expression | journal = mSphere | volume = 2 | issue = 2 | pages = e00385–16 | year = 2017 | pmid = 28435892 | pmc = 5397569 | doi = 10.1128/mSphere.00385-16 }} zebrafish (Danio rerio),{{cite journal | vauthors = Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK | title = Efficient genome editing in zebrafish using a CRISPR-Cas system | journal = Nature Biotechnology | volume = 31 | issue = 3 | pages = 227–229 | date = March 2013 | pmid = 23360964 | pmc = 3686313 | doi = 10.1038/nbt.2501 }} fruit flies (Drosophila melanogaster),{{cite journal | vauthors = Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O'Connor-Giles KM | title = Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease | journal = Genetics | volume = 194 | issue = 4 | pages = 1029–1035 | date = August 2013 | pmid = 23709638 | pmc = 3730909 | doi = 10.1534/genetics.113.152710 }}{{cite journal | vauthors = Bassett AR, Tibbit C, Ponting CP, Liu JL | title = Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system | journal = Cell Reports | volume = 4 | issue = 1 | pages = 220–228 | date = July 2013 | pmid = 23827738 | pmc = 3714591 | doi = 10.1016/j.celrep.2013.06.020 }} ants (Harpegnathos saltator{{cite journal | vauthors = Yan H, Opachaloemphan C, Mancini G, Yang H, Gallitto M, Mlejnek J, Leibholz A, Haight K, Ghaninia M, Huo L, Perry M, Slone J, Zhou X, Traficante M, Penick CA, Dolezal K, Gokhale K, Stevens K, Fetter-Pruneda I, Bonasio R, Zwiebel LJ, Berger SL, Liebig J, Reinberg D, Desplan C | title = An Engineered orco Mutation Produces Aberrant Social Behavior and Defective Neural Development in Ants | journal = Cell | volume = 170 | issue = 4 | pages = 736–747.e9 | date = August 2017 | pmid = 28802043 | pmc = 5587193 | doi = 10.1016/j.cell.2017.06.051 }} and Ooceraea biroi{{cite journal | vauthors = Trible W, Olivos-Cisneros L, McKenzie SK, Saragosti J, Chang NC, Matthews BJ, Oxley PR, Kronauer DJ | title = orco Mutagenesis Causes Loss of Antennal Lobe Glomeruli and Impaired Social Behavior in Ants | journal = Cell | volume = 170 | issue = 4 | pages = 727–735.e10 | date = August 2017 | pmid = 28802042 | pmc = 5556950 | doi = 10.1016/j.cell.2017.07.001 }}), mosquitoes (Aedes aegypti{{cite journal | vauthors = Kistler KE, Vosshall LB, Matthews BJ | title = Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti | journal = Cell Reports | volume = 11 | issue = 1 | pages = 51–60 | date = April 2015 | pmid = 25818303 | pmc = 4394034 | doi = 10.1016/j.celrep.2015.03.009 }}), nematodes (Caenorhabditis elegans),{{cite journal | vauthors = Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA | title = Heritable genome editing in C. elegans via a CRISPR-Cas9 system | journal = Nature Methods | volume = 10 | issue = 8 | pages = 741–743 | date = August 2013 | pmid = 23817069 | pmc = 3822328 | doi = 10.1038/nmeth.2532 }} plants,{{cite journal | vauthors = Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP | title = Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice | journal = Nucleic Acids Research | volume = 41 | issue = 20 | pages = e188 | date = November 2013 | pmid = 23999092 | pmc = 3814374 | doi = 10.1093/nar/gkt780 }} mice (Mus musculus domesticus),{{cite journal | vauthors = Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R | author-link6=Feng Zhang | title = One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering | journal = Cell | volume = 153 | issue = 4 | pages = 910–918 | date = May 2013 | pmid = 23643243 | pmc = 3969854 | doi = 10.1016/j.cell.2013.04.025 }}{{cite journal | vauthors = Soni D, Wang DM, Regmi SC, Mittal M, Vogel SM, Schlüter D, Tiruppathi C | title = Deubiquitinase function of A20 maintains and repairs endothelial barrier after lung vascular injury | journal = Cell Death Discovery | volume = 4 | issue = 60 | page = 60| date = May 2018 | pmid = 29796309| pmc =5955943 | doi = 10.1038/s41420-018-0056-3}} monkeys{{cite journal | vauthors = Guo X, Li XJ | title = Targeted genome editing in primate embryos | journal = Cell Research | volume = 25 | issue = 7 | pages = 767–768 | date = July 2015 | pmid = 26032266 | pmc = 4493275 | doi = 10.1038/cr.2015.64 }} and human embryos.{{cite journal | vauthors = Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M, Greely HT, Jinek M, Martin GS, Penhoet E, Puck J, Sternberg SH, Weissman JS, Yamamoto KR | author-link9=Jennifer Doudna | title = Biotechnology. A prudent path forward for genomic engineering and germline gene modification | journal = Science | volume = 348 | issue = 6230 | pages = 36–38 | date = April 2015 | pmid = 25791083 | pmc = 4394183 | doi = 10.1126/science.aab1028 | bibcode = 2015Sci...348...36B }}

CRISPR has been modified to make programmable transcription factors that allow activation or silencing of targeted genes.{{cite journal | vauthors = Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS | title = CRISPR interference (CRISPRi) for sequence-specific control of gene expression | journal = Nature Protocols | volume = 8 | issue = 11 | pages = 2180–2196 | date = November 2013|pmc=3922765| pmid = 24136345 | doi = 10.1038/nprot.2013.132 }}

File:Cas12a vs Cas9 cleavage position.svg and Cas9 with the position of DNA cleavage shown relative to their PAM sequences in a zoom-in]]

The CRISPR-Cas9 system has been shown to make effective gene edits in Human tripronuclear zygotes, as first described in a 2015 paper by Chinese scientists P. Liang and Y. Xu. The system made a successful cleavage of mutant Beta-Hemoglobin (HBB) in 28 out of 54 embryos. Four out of the 28 embryos were successfully recombined using a donor template. The scientists showed that during DNA recombination of the cleaved strand, the homologous endogenous sequence HBD competes with the exogenous donor template. DNA repair in human embryos is much more complicated and particular than in derived stem cells.{{cite journal |vauthors=Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J | title = CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes | journal = Protein & Cell | volume = 6 | issue = 5 | pages = 363–372 | date = May 2015 | doi = 10.1007/s13238-015-0153-5 | pmid=25894090 | pmc=4417674}}

{{main|Cas12a}}

Cas12a, a Class II Type V CRISPR-associated nuclease, was characterized in 2015 and was formerly known as Cpf1 . This nuclease is found in the CRISPR-Cpf1 system of bacteria such as Francisella novicida . 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 ; 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 ; and it requires only a CRISPR RNA (crRNA) for effective targeting, whereas Cas9 necessitates both a crRNA and a trans-activating crRNA (tracrRNA) .

These differences may confer certain advantages to Cas12a over Cas9. For instance, the smaller size of Cas12a's crRNAs makes them well-suited for multiplexed genome editing, allowing for a greater number of guide RNAs to be packaged into a single vector compared to Cas9's single guide RNAs (sgRNAs) . The sticky 5′ overhangs generated by Cas12a can also be utilized for DNA assembly with higher target specificity than traditional restriction enzyme cloning. Furthermore, Cas12a cleaves DNA 18–23 base pairs downstream from the PAM site. This characteristic ensures that the recognition sequence remains intact after repair, enabling Cas12a to perform multiple rounds of DNA cleavage. Conversely, Cas9 cleaves only 3 base pairs upstream of the PAM site, leading to indel mutations from the non-homologous end joining (NHEJ) pathway that can disrupt the recognition sequence, thereby preventing subsequent rounds of cutting. A notable feature of Cas12a, in contrast to Cas9, is its ability to remain bound to its target DNA after cleavage and subsequently cleave other single-stranded DNA molecules non-discriminately . This property, termed "collateral cleavage" or "trans-cleavage" activity, has been harnessed for the development of various diagnostic technologies .

Cas12a Structural and Functional Comparison with Cas9

While classified as a Class II Type V CRISPR-associated nuclease, Cas12a lacks the NHN endonuclease domain found in Cas9 and instead relies on its RuvC-like domain for sequential DNA cleavage, resulting in the production of staggered ends . Unlike Cas9, Cas12a possesses a unique zinc finger-like domain, which may contribute to DNA binding or structural stability. A significant advantage of Cas12a over Cas9 is its inherent RNase activity within the WED domain, enabling the self-processing of precursor crRNAs (pre-crRNAs) into mature crRNAs . This eliminates the requirement for a trans-activating crRNA (tracrRNA), simplifying guide RNA design and facilitating multiplexed editing through the packaging of multiple crRNAs into a single vector.

Cas12a Multiplex Genome Editing

Multiplex genome editing is a powerful technique that allows for the simultaneous editing of multiple genes. Cas12a is particularly useful in this context due to features that simplify its application compared to Cas9 . For instance, Cas12a requires only a short CRISPR RNA (crRNA) to target and edit genes . These crRNAs can be easily multiplexed by packaging them together into a single delivery system, enabling the simultaneous editing of multiple genomic loci .

Furthermore, Cas12a and Cas9 exhibit differences in their protospacer adjacent motif (PAM) requirements. While Cas9 typically looks for G-rich sequences, Cas12a targets T-rich sequences. This difference expands the range of targetable genomic sites, allowing Cas12a to edit genes in regions where Cas9 may not be efficient, such as AT-rich areas prevalent in plant genomes , providing greater flexibility for researchers.

The ability of Cas12a to deliver its own crRNA (CRISPR RNA) without the need for additional components like the tracrRNA required by Cas9 further simplifies the design and delivery of guide RNAs for multiplexed editing.

In 2016, the nuclease {{visible anchor|Cas13a}} (formerly known as {{visible anchor|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.{{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 }} This collateral cleavage property has been exploited for the development of various diagnostic technologies.{{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 }}{{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 }}{{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 }}

In 2018, a team led by Silvana Konermann and Patrick Hsu at the Salk Institute reported the discovery of Cas13d, a new subclass of compact RNA-targeting CRISPR effectors. An engineered variant of Ruminococcus flavefaciens Cas13d, termed CasRx, exhibited robust activity in human cells, with high efficiency and specificity relative to RNA interference, and could be packaged into adeno-associated virus (AAV) for applications in transcriptome engineering and gene therapy.{{cite journal | vauthors = Konermann S, Lotfy P, Brideau NJ, Oki J, Shokhirev MN, Hsu PD | title = Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors | journal = Cell | date = April 2018 | pmid = 29551272 | pmc = 5910255 | doi = 10.1016/j.cell.2018.02.033 }}

In 2021, researchers in China characterized novel miniature Cas13 protein (mCas13) variants, Cas13X and Cas13Y. Using a small portion of N gene sequence from SARS-CoV-2 as a target in characterization of mCas13, revealed the sensitivity and specificity of mCas13 coupled with RT-LAMP for detection of SARS-CoV-2 in both synthetic and clinical samples over other available standard tests like RT-qPCR (1 copy/μL).{{cite journal | vauthors = Mahas A, Wang Q, Marsic T, Mahfouz MM | title = A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics | journal = ACS Synthetic Biology | volume = 10 | issue = 10 | pages = 2541–2551 | date = October 2021 | pmid = 34546709 | pmc = 8482783 | doi = 10.1021/acssynbio.1c00181 }}

The CRISPR array is made up of an AT-rich leader sequence followed by short repeats that are separated by unique spacers.{{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}} CRISPR repeats typically range in size from 28 to 37 base pairs (bps), though there can be as few as 23 bp and as many as 55 bp.{{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 }} Some show dyad symmetry, implying the formation of a 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). New spacers can appear rapidly as part of the immune response to phage infection. There are usually fewer than 50 units of the repeat-spacer sequence in a CRISPR array.

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].

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.{{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 }}

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.{{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 }} The 6 system types are divided into 33 subtypes.{{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 }} 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,{{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 }} but exceptions exist due to module shuffling. Many organisms contain multiple CRISPR-Cas systems suggesting that they are compatible and may share components.{{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 }}{{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 = }} 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}}

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File:The Stages of CRISPR immunity.svg and Cas2 and cleavage of a protospacer.
(2) The protospacer is ligated to the direct repeat adjacent to the leader sequence and
(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.
(4) The primary CRISPR transcript is cleaved by cas genes to produce crRNAs.
(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.
(6) In type II and type III systems secondary trimming is performed at either the 5' or 3' end to produce mature crRNAs.
(7) Mature crRNAs associate with Cas proteins to form interference complexes.
(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.]]

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File:13 Hegasy CRISPR pre crRNA Wiki E CCBYSA.png

File:14 Hegasy Cas9 3D Complex Wiki E CCBYSA.png

File:15 Hegasy Cas9 DNA Tool Wiki E CCBYSA.png

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CRISPR-Cas immunity is a natural process of bacteria and archaea.{{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 }} CRISPR-Cas prevents bacteriophage infection, conjugation and natural transformation by degrading foreign nucleic acids that enter the cell.{{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 }}

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.{{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 }}{{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 }}{{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 }}{{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 }}{{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 }}

Multiple Cas1 proteins have been characterised and their structures resolved.{{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 }}{{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 }}{{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 }} 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. Representative Cas2 proteins have been characterised and possess either (single strand) ssRNA-{{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 }} or (double strand) dsDNA-{{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 }}{{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 }} 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.{{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 }} In this complex, Cas2 performs a non-enzymatic scaffolding role, binding double-stranded fragments of invading DNA, while Cas1 binds the single-stranded flanks of the DNA and catalyses their integration into CRISPR arrays.{{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 }}{{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 }}{{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 }} New spacers are usually added at the beginning of the CRISPR next to the leader sequence creating a chronological record of viral infections.{{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 }} In E. coli a histone like protein called integration host factor (IHF), which binds to the leader sequence, is responsible for the accuracy of this integration.{{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 }} IHF also enhances integration efficiency in the type I-F system of Pectobacterium atrosepticum,{{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 }} but in other systems, different host factors may be required{{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 }}

{{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 motifs (PAM). Analysis of CRISPR-Cas systems showed PAMs to be important for type I and type II, but not type III systems during acquisition.{{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 }}{{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 }}{{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 }}{{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 }}{{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 }} 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.{{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 }}{{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 }} The conservation of the PAM sequence differs between CRISPR-Cas systems and appears to be evolutionarily linked to Cas1 and the leader sequence.{{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 }}

New spacers are added to a CRISPR array in a directional manner, occurring preferentially,{{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 }}{{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 = 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 }} but not exclusively, adjacent 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.

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.{{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 }}{{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 }} 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. 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.

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.

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. This observation led to the hypothesis that the acquisition machinery slides along the foreign DNA after priming to find a new protospacer.

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. 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{{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 }}{{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 }}{{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 }} created by the pairing of identical repeats that flank the crRNA.{{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 }} 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.{{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 }}{{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 }}{{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 }}

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). 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.

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.{{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 }}{{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 }}{{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 }} The type I-E complex (commonly referred to as Cascade) requires five Cas proteins bound to a single crRNA.{{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 }}{{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}}

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. 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.{{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 }}{{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 }} The type III systems analysed from S. solfataricus and P. furiosus both target the mRNA of phages rather than phage DNA genome, which may make these systems uniquely capable of targeting RNA-based phage genomes. Type III systems were also found to target DNA in addition to RNA using a different Cas protein in the complex, Cas10.{{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 }} The DNA cleavage was shown to be transcription dependent.{{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 }}

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.{{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 }} RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

{{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.{{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 }} 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.{{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 }} The evolution of these various class 1 effector module cas genes was guided by various mechanisms, such as duplication events.{{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 }} On the other hand, each type of class 2 effector module arose from subsequent independent insertions of mobile genetic elements.{{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 }} 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. 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.{{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 }} 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.{{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 }}

CRISPR-Cas can immunize bacteria against certain phages and thus halt transmission. For this reason, Koonin described CRISPR-Cas as a Lamarckian inheritance mechanism.{{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 }} 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".{{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 }} 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.{{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 }} On the other hand, the evolution of the Cas gene machinery that facilitates the system evolves through classic Darwinian evolution.

Analysis of CRISPR sequences revealed coevolution of host and viral genomes.{{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 }}

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. Similar stringency is required in PAM or the bacterial strain remains phage sensitive.

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. Some CRISPR loci evolve more rapidly than others, which allowed the strains' phylogenetic relationships to be determined. A comparative genomic analysis showed that E. coli and 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.{{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 }}

Metagenomic analysis of two acid-mine-drainage biofilms 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. 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.

From the same environment, a single strain was tracked using 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, 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.{{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 }} 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.

CRISPR evolution was studied in chemostats 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.{{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 }} During the same interval, the phage developed single-nucleotide polymorphisms that became fixed in the population, suggesting that targeting had prevented phage replication absent these mutations.

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.{{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 }} The chemostat and observational studies suggest many nuances to CRISPR and phage (co)evolution.

CRISPRs are widely distributed among bacteria and archaea and show some sequence similarities. 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.{{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 }}

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.{{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 }}{{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 }}{{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 }}{{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 }} 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.

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{{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 }} or by using direct repeat sequences in partially assembled CRISPR arrays from contigs (overlapping DNA segments that together represent a consensus region of DNA) and direct repeat sequences from published genomes{{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 }} as a hook for identifying direct repeats in individual reads.

Another way for bacteria to defend against phage infection is by having 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.{{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 }} 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.{{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}} 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.{{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}}

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 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.{{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 }}{{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 }}

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.

{{main|CRISPR gene editing}}

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 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.{{Cite journal |last=Anzalone |first=Andrew V. |last2=Koblan |first2=Luke W. |last3=Liu |first3=David R. |date=July 2020 |title=Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors |url=https://www.nature.com/articles/s41587-020-0561-9 |journal=Nature Biotechnology |language=en |volume=38 |issue=7 |pages=824–844 |doi=10.1038/s41587-020-0561-9 |issn=1546-1696}} It makes use of the cell's natural DNA repair systems, including non-homologous end joining, homology-directed repair, or mismatch repair, to modify, insert, or delete genetic material at these specific cut sites. {{Cite journal |last=Anzalone |first=Andrew V. |last2=Randolph |first2=Peyton B. |last3=Davis |first3=Jessie R. |last4=Sousa |first4=Alexander A. |last5=Koblan |first5=Luke W. |last6=Levy |first6=Jonathan M. |last7=Chen |first7=Peter J. |last8=Wilson |first8=Christopher |last9=Newby |first9=Gregory A. |last10=Raguram |first10=Aditya |last11=Liu |first11=David R. |date=December 2019 |title=Search-and-replace genome editing without double-strand breaks or donor DNA |url=https://www.nature.com/articles/s41586-019-1711-4 |journal=Nature |language=en |volume=576 |issue=7785 |pages=149–157 |doi=10.1038/s41586-019-1711-4 |issn=1476-4687|pmc=6907074 }} This technology has transformed fields such as genetics, medicine,{{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 }}{{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 | doi = 10.1186/s12967-024-05570-4 | doi-access = free | pmc = 11312220 }}{{cite journal |vauthors=Kaur R, Gupta S, Chauhan A, Mishra V, Sharma MK, Singh J |date=August 2024 |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 |doi=10.1007/s11033-024-09840-8 |pmid=39115550}} and agriculture,{{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 | date = July 2024 | page = 1884 | pmid = 39065411 | pmc = 11279650 | doi = 10.3390/plants13141884 | doi-access = free | bibcode = 2024Plnts..13.1884M }} 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.{{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 | issue = | pages = 103–110 | date = 2018 | pmid = 28879860 | doi = 10.21775/cimb.026.103 | doi-access = free }}{{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 | doi = 10.1161/ATVBAHA.123.318069 | pmc = 10948015 }}

{{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 documentary film)
• MAGESTIC
• New eugenics
• Prime editing
• RNAi
• SiRNA
• Surveyor nuclease assay
• Synthetic biology
• Zinc finger

{{div col end}}

{{Reflist|group=Note}}

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{{reflist}}

{{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}}

{{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|date=September 9, 2020|season=47|number=9|url=https://www.pbs.org/wgbh/nova/video/human-nature/|access-date=April 7, 2023}}

• {{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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