Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering
2016; Cell Press; Volume: 164; Issue: 1-2 Linguagem: Inglês
10.1016/j.cell.2015.12.035
ISSN1097-4172
AutoresAddison V. Wright, James K. Nuñez, Jennifer A. Doudna,
Tópico(s)Genetically Modified Organisms Research
ResumoBacteria and archaea possess a range of defense mechanisms to combat plasmids and viral infections. Unique among these are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems, which provide adaptive immunity against foreign nucleic acids. CRISPR systems function by acquiring genetic records of invaders to facilitate robust interference upon reinfection. In this Review, we discuss recent advances in understanding the diverse mechanisms by which Cas proteins respond to foreign nucleic acids and how these systems have been harnessed for precision genome manipulation in a wide array of organisms. Bacteria and archaea possess a range of defense mechanisms to combat plasmids and viral infections. Unique among these are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems, which provide adaptive immunity against foreign nucleic acids. CRISPR systems function by acquiring genetic records of invaders to facilitate robust interference upon reinfection. In this Review, we discuss recent advances in understanding the diverse mechanisms by which Cas proteins respond to foreign nucleic acids and how these systems have been harnessed for precision genome manipulation in a wide array of organisms. CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) adaptive immune systems are found in roughly 50% of bacteria and 90% of archaea (Makarova et al., 2015Makarova K.S. Wolf Y.I. Alkhnbashi O.S. Costa F. Shah S.A. Saunders S.J. Barrangou R. Brouns S.J.J. Charpentier E. Haft D.H. et al.An updated evolutionary classification of CRISPR-Cas systems.Nat. Rev. Microbiol. 2015; 13: 722-736Crossref PubMed Scopus (46) Google Scholar). These systems function alongside restriction-modification systems, abortive infections, and adsorption blocks to defend prokaryotic populations against phage infection (Labrie et al., 2010Labrie S.J. Samson J.E. Moineau S. Bacteriophage resistance mechanisms.Nat. Rev. Microbiol. 2010; 8: 317-327Crossref PubMed Scopus (427) Google Scholar). Unlike other mechanisms of cellular defense, which provide generalized protection against any invaders not possessing countermeasures, CRISPR immunity functions analogously to vertebrate adaptive immunity by generating records of previous infections to elicit a rapid and robust response upon reinfection. CRISPR-Cas systems are generally defined by a genomic locus called the CRISPR array, a series of ∼20–50 base-pair (bp) direct repeats separated by unique “spacers” of similar length and preceded by an AT-rich “leader” sequence (Jansen et al., 2002Jansen R. Embden J.D. Gaastra W. Schouls L.M. 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Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product.J. Bacteriol. 1987; 169: 5429-5433Crossref PubMed Google Scholar, Mojica et al., 2005Mojica F.J. Díez-Villaseñor C. García-Martínez J. Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.J. Mol. Evol. 2005; 60: 174-182Crossref PubMed Scopus (402) Google Scholar, Pourcel et al., 2005Pourcel C. Salvignol G. Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.Microbiology. 2005; 151: 653-663Crossref PubMed Scopus (342) Google Scholar). Sequences in foreign DNA matching spacers are referred to as “protospacers.” In 2007, it was shown that a spacer matching a phage genome immunizes the host microbe against the corresponding phage and that infection by a novel phage leads to the expansion of the CRISPR array by addition of new spacers originating from the phage genome (Barrangou et al., 2007Barrangou R. Fremaux C. Deveau H. Richards M. Boyaval P. Moineau S. Romero D.A. Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes.Science. 2007; 315: 1709-1712Crossref PubMed Scopus (1145) Google Scholar). CRISPR immunity is divided into three stages: spacer acquisition, CRISPR RNA (crRNA) biogenesis, and interference (Figure 1A) (Makarova et al., 2011bMakarova K.S. Haft D.H. Barrangou R. Brouns S.J. Charpentier E. Horvath P. Moineau S. Mojica F.J. Wolf Y.I. Yakunin A.F. et al.Evolution and classification of the CRISPR-Cas systems.Nat. Rev. Microbiol. 2011; 9: 467-477Crossref PubMed Scopus (647) Google Scholar, van der Oost et al., 2009van der Oost J. Jore M.M. Westra E.R. Lundgren M. Brouns S.J. CRISPR-based adaptive and heritable immunity in prokaryotes.Trends Biochem. Sci. 2009; 34: 401-407Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). During spacer acquisition, also known as adaptation, foreign DNA is identified, processed, and integrated into the CRISPR locus as a new spacer. The crRNA biogenesis or expression stage involves CRISPR locus transcription, often as a single pre-crRNA, and its subsequent processing into mature crRNAs that each contain a single spacer. In the interference stage, an effector complex uses the crRNA to identify and destroy any phage or plasmid bearing sequence complementarity to the spacer sequence of the crRNA. These steps are carried out primarily by Cas proteins, which are encoded by cas genes flanking the CRISPR arrays. The specific complement of cas genes varies widely. CRISPR-Cas systems can be classified based on the presence of “signature genes” into six types, which are additionally grouped into two classes (Figure 1B) (Makarova et al., 2011bMakarova K.S. Haft D.H. Barrangou R. Brouns S.J. Charpentier E. Horvath P. Moineau S. Mojica F.J. Wolf Y.I. Yakunin A.F. et al.Evolution and classification of the CRISPR-Cas systems.Nat. Rev. Microbiol. 2011; 9: 467-477Crossref PubMed Scopus (647) Google Scholar, Makarova et al., 2015Makarova K.S. Wolf Y.I. Alkhnbashi O.S. Costa F. Shah S.A. Saunders S.J. Barrangou R. Brouns S.J.J. Charpentier E. Haft D.H. et al.An updated evolutionary classification of CRISPR-Cas systems.Nat. Rev. Microbiol. 2015; 13: 722-736Crossref PubMed Scopus (46) Google Scholar, Shmakov et al., 2015Shmakov S. Abudayyeh O.O. Makarova K.S. Wolf Y.I. Gootenberg J.S. Semenova E. Minakhin L. Joung J. Konermann S. Severinov K. et al.Discovery and functional characterization of diverse Class 2 CRISPR-Cas systems.Mol. Cell. 2015; 60: 385-397Abstract Full Text Full Text PDF PubMed Google Scholar). Types I–III are the best studied, while Types IV–VI have only recently been identified (Makarova and Koonin, 2015Makarova K.S. Koonin E.V. Annotation and Classification of CRISPR-Cas Systems.Methods Mol. Biol. 2015; 1311: 47-75Crossref PubMed Google Scholar, Makarova et al., 2015Makarova K.S. Wolf Y.I. Alkhnbashi O.S. Costa F. Shah S.A. Saunders S.J. Barrangou R. Brouns S.J.J. Charpentier E. Haft D.H. et al.An updated evolutionary classification of CRISPR-Cas systems.Nat. Rev. Microbiol. 2015; 13: 722-736Crossref PubMed Scopus (46) Google Scholar, Shmakov et al., 2015Shmakov S. Abudayyeh O.O. Makarova K.S. Wolf Y.I. Gootenberg J.S. Semenova E. Minakhin L. Joung J. Konermann S. Severinov K. et al.Discovery and functional characterization of diverse Class 2 CRISPR-Cas systems.Mol. Cell. 2015; 60: 385-397Abstract Full Text Full Text PDF PubMed Google Scholar). The signature protein of Type I systems is Cas3, a protein with nuclease and helicase domains that functions in foreign DNA degradation to cleave DNA that is recognized by the multi-protein-crRNA complex Cascade (CRISPR-associated complex for antiviral defense). In Type II systems, the signature cas9 gene encodes the sole protein necessary for interference. Type III systems are signified by Cas10, which assembles into a Cascade-like interference complex for target search and destruction. Type IV systems have Csf1, an uncharacterized protein proposed to form part of a Cascade-like complex, though these systems are often found as isolated cas genes without an associated CRISPR array (Makarova and Koonin, 2015Makarova K.S. Koonin E.V. Annotation and Classification of CRISPR-Cas Systems.Methods Mol. Biol. 2015; 1311: 47-75Crossref PubMed Google Scholar). Type V systems also contain a Cas9-like single nuclease, either Cpf1, C2c1, or C2c3, depending on the subtype (Shmakov et al., 2015Shmakov S. Abudayyeh O.O. Makarova K.S. Wolf Y.I. Gootenberg J.S. Semenova E. Minakhin L. Joung J. Konermann S. Severinov K. et al.Discovery and functional characterization of diverse Class 2 CRISPR-Cas systems.Mol. Cell. 2015; 60: 385-397Abstract Full Text Full Text PDF PubMed Google Scholar, Zetsche et al., 2015aZetsche B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. Essletzbichler P. Volz S.E. Joung J. van der Oost J. Regev A. et al.Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System.Cell. 2015; 163: 759-771Abstract Full Text Full Text PDF PubMed Google Scholar). Type VI systems have C2c2, a large protein with two predicted HEPN (higher eukaryotes and prokaryotes nucleotide-binding) RNase domains (Shmakov et al., 2015Shmakov S. Abudayyeh O.O. Makarova K.S. Wolf Y.I. Gootenberg J.S. Semenova E. Minakhin L. Joung J. Konermann S. Severinov K. et al.Discovery and functional characterization of diverse Class 2 CRISPR-Cas systems.Mol. Cell. 2015; 60: 385-397Abstract Full Text Full Text PDF PubMed Google Scholar). Type I, III, and IV systems are considered Class 1 systems based on their multi-subunit effector complexes, while the single-subunit effector Type II, V, and VI systems are grouped into Class 2 (Makarova et al., 2015Makarova K.S. Wolf Y.I. Alkhnbashi O.S. Costa F. Shah S.A. Saunders S.J. Barrangou R. Brouns S.J.J. Charpentier E. Haft D.H. et al.An updated evolutionary classification of CRISPR-Cas systems.Nat. Rev. Microbiol. 2015; 13: 722-736Crossref PubMed Scopus (46) Google Scholar, Shmakov et al., 2015Shmakov S. Abudayyeh O.O. Makarova K.S. Wolf Y.I. Gootenberg J.S. Semenova E. Minakhin L. Joung J. Konermann S. Severinov K. et al.Discovery and functional characterization of diverse Class 2 CRISPR-Cas systems.Mol. Cell. 2015; 60: 385-397Abstract Full Text Full Text PDF PubMed Google Scholar). The study of CRISPR biology has revealed enzyme mechanisms that can be harnessed for precision genome engineering and other applications, leading to an explosion of interest in both native CRISPR pathways and the use of these systems for applications in animals, plants, microbes, and humans. In this Review, we discuss recent advancements in the field that reveal unexpected divergence, as well as unifying themes underlying the three stages of CRISPR immunity. In each case, we highlight the ways in which these systems are being harnessed for applications across many areas of biology. CRISPR immunity begins with the detection and integration of foreign DNA into the host cell’s chromosome. In the Streptococcus thermophilus Type II-A system, where acquisition was first detected experimentally, new spacers from bacteriophage DNA are inserted into the leader end of the CRISPR locus, causing duplication of the first repeat to maintain the repeat-spacer architecture (Figure 1A) (Barrangou et al., 2007Barrangou R. Fremaux C. Deveau H. Richards M. Boyaval P. Moineau S. Romero D.A. Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes.Science. 2007; 315: 1709-1712Crossref PubMed Scopus (1145) Google Scholar). Subsequent studies using the E. coli Type I-E system verified that Cas1 and Cas2 mediate spacer acquisition (Datsenko et al., 2012Datsenko K.A. Pougach K. Tikhonov A. Wanner B.L. Severinov K. Semenova E. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system.Nat. Commun. 2012; 3: 945Crossref PubMed Scopus (154) Google Scholar, Swarts et al., 2012Swarts D.C. Mosterd C. van Passel M.W. Brouns S.J. CRISPR interference directs strand specific spacer acquisition.PLoS ONE. 2012; 7: e35888Crossref PubMed Scopus (120) Google Scholar, Yosef et al., 2012Yosef I. Goren M.G. Qimron U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli.Nucleic Acids Res. 2012; 40: 5569-5576Crossref PubMed Scopus (168) Google Scholar). The selection of new protospacer sequences is nonrandom and, in most systems, depends on the presence of a 2–5 nucleotide protospacer adjacent motif (PAM) found next to the protospacer sequence (Deveau et al., 2008Deveau H. Barrangou R. Garneau J.E. Labonté J. Fremaux C. Boyaval P. Romero D.A. Horvath P. Moineau S. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus.J. Bacteriol. 2008; 190: 1390-1400Crossref PubMed Scopus (364) Google Scholar, Mojica et al., 2009Mojica F.J. Díez-Villaseñor C. García-Martínez J. Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system.Microbiology. 2009; 155: 733-740Crossref PubMed Scopus (306) Google Scholar). PAM-specific selection of protospacers is critical for immunity, as crRNA-guided interference in most systems depends on the PAM sequence for foreign DNA detection and destruction, which avoids self-targeting at the PAM-free CRISPR locus. Interestingly, spacers originating from the host genome are present in almost 20% of CRISPR-containing organisms, suggesting alternative roles of the CRISPR-Cas machinery in directing other processes such as endogenous gene regulation and genome evolution (Westra et al., 2014Westra E.R. Buckling A. Fineran P.C. CRISPR-Cas systems: beyond adaptive immunity.Nat. Rev. Microbiol. 2014; 12: 317-326Crossref PubMed Scopus (38) Google Scholar). Spacer acquisition has been observed experimentally in various systems across Types I–III. Here, we focus on recent mechanistic studies of acquisition in Type I-E and Type II-A systems, in which the most comprehensive studies have been done. Acquisition in E. coli occurs via two mechanisms—naive and primed (Figure 2A). Naive acquisition initiates upon infection by previously unencountered DNA and relies on the Cas1-Cas2 integrase complex to recognize and acquire new spacers from foreign DNA. Overexpression of Cas1 and Cas2 in the absence of other Cas proteins leads to the acquisition of 33 bp spacers at the leader-proximal end of the CRISPR array (Datsenko et al., 2012Datsenko K.A. Pougach K. Tikhonov A. Wanner B.L. Severinov K. Semenova E. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system.Nat. Commun. 2012; 3: 945Crossref PubMed Scopus (154) Google Scholar, Yosef et al., 2012Yosef I. Goren M.G. Qimron U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli.Nucleic Acids Res. 2012; 40: 5569-5576Crossref PubMed Scopus (168) Google Scholar). The PAM of the E. coli CRISPR-Cas system was identified as 5′-AWG-3′, with the G becoming the first nucleotide of the integrated spacer (Datsenko et al., 2012Datsenko K.A. Pougach K. Tikhonov A. Wanner B.L. Severinov K. Semenova E. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system.Nat. Commun. 2012; 3: 945Crossref PubMed Scopus (154) Google Scholar, Díez-Villaseñor et al., 2013Díez-Villaseñor C. Guzmán N.M. Almendros C. García-Martínez J. Mojica F.J. CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli.RNA Biol. 2013; 10: 792-802Crossref PubMed Scopus (34) Google Scholar, Levy et al., 2015Levy A. Goren M.G. Yosef I. Auster O. Manor M. Amitai G. Edgar R. Qimron U. Sorek R. 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CRISPR interference directs strand specific spacer acquisition.PLoS ONE. 2012; 7: e35888Crossref PubMed Scopus (120) Google Scholar, Yosef et al., 2012Yosef I. Goren M.G. Qimron U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli.Nucleic Acids Res. 2012; 40: 5569-5576Crossref PubMed Scopus (168) Google Scholar, Yosef et al., 2013Yosef I. Shitrit D. Goren M.G. Burstein D. Pupko T. Qimron U. DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array.Proc. Natl. Acad. Sci. USA. 2013; 110: 14396-14401Crossref PubMed Scopus (23) Google Scholar). In addition to the PAM, a dinucleotide motif, AA, found at the 3′ end of the protospacer was also shown to be present in a disproportionately large number of spacers (Yosef et al., 2013Yosef I. Shitrit D. Goren M.G. Burstein D. Pupko T. Qimron U. DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array.Proc. Natl. Acad. Sci. 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CRISPR interference directs strand specific spacer acquisition.PLoS ONE. 2012; 7: e35888Crossref PubMed Scopus (120) Google Scholar). Priming increases the host’s repertoire of functional spacers, allowing the host to adapt to invaders that evade the CRISPR-Cas system by mutation. Cascade is capable of binding escape mutant target sites, and recent single-molecule studies showed that the presence of Cas1 and Cas2 allows for the recruitment of Cas3 to these sites (Blosser et al., 2015Blosser T.R. Loeff L. Westra E.R. Vlot M. Künne T. Sobota M. Dekker C. Brouns S.J. Joo C. Two distinct DNA binding modes guide dual roles of a CRISPR-Cas protein complex.Mol. Cell. 2015; 58: 60-70Abstract Full Text Full Text PDF PubMed Google Scholar, Redding et al., 2015Redding S. Sternberg S.H. Marshall M. Gibb B. Bhat P. Guegler C.K. Wiedenheft B. Doudna J.A. Greene E.C. Surveillance and processing of foreign DNA by the Escherichia coli CRISPR-Cas system.Cell. 2015; 163: 854-865Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, Richter et al., 2014Richter C. Dy R.L. McKenzie R.E. Watson B.N. Taylor C. Chang J.T. McNeil M.B. Staals R.H. Fineran P.C. Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.Nucleic Acids Res. 2014; 42: 8516-8526Crossref PubMed Scopus (25) Google Scholar). The recruited Cas3 can then translocate in either direction, in contrast to the unidirectional movement observed at perfect targets, without degrading the target DNA (Redding et al., 2015Redding S. Sternberg S.H. Marshall M. Gibb B. Bhat P. Guegler C.K. Wiedenheft B. Doudna J.A. Greene E.C. Surveillance and processing of foreign DNA by the Escherichia coli CRISPR-Cas system.Cell. 2015; 163: 854-865Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Cas1 and Cas2 may accompany the translocating Cas3 and be activated for protospacer selection, allowing for robust acquisition on either side of the target site. Primed acquisition has also been shown experimentally in the P. atrosepticum Type I–F system, in which Cas2 and Cas3 are naturally fused as a single polypeptide that associates with Cas1, as well as in the Haloarcula hispanica Type I-B system, where naive acquisition was not experimentally observed (Li et al., 2014Li M. Wang R. Zhao D. Xiang H. Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process.Nucleic Acids Res. 2014; 42: 2483-2492Crossref PubMed Scopus (32) Google Scholar, Richter et al., 2014Richter C. Dy R.L. McKenzie R.E. Watson B.N. Taylor C. Chang J.T. McNeil M.B. Staals R.H. Fineran P.C. Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.Nucleic Acids Res. 2014; 42: 8516-8526Crossref PubMed Scopus (25) Google Scholar, Richter et al., 2012Richter C. Gristwood T. Clulow J.S. Fineran P.C. In vivo protein interactions and complex formation in the Pectobacterium atrosepticum subtype I-F CRISPR/Cas System.PLoS ONE. 2012; 7: e49549Crossref PubMed Scopus (19) Google Scholar). Acquisition in H. hispanica also requires Cas4, a 5′→3′ exonuclease found in most Type I subtypes as well as Type II-B and Type V systems, and which might be involved in generating 3′ overhangs on protospacers prior to integration (Lemak et al., 2013Lemak S. Beloglazova N. Nocek B. Skarina T. Flick R. Brown G. Popovic A. Joachimiak A. Savchenko A. Yakunin A.F. Toroidal structure and DNA cleavage by the CRISPR-associated [4Fe-4S] cluster containing Cas4 nuclease SSO0001 from Sulfolobus solfataricus.J. Am. Chem. Soc. 2013; 135: 17476-17487Crossref PubMed Scopus (10) Google Scholar, Li et al., 2014Li M. Wang R. Zhao D. Xiang H. Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process.Nucleic Acids Res. 2014; 42: 2483-2492Crossref PubMed Scopus (32) Google Scholar, Makarova et al., 2015Makarova K.S. Wolf Y.I. Alkhnbashi O.S. Costa F. Shah S.A. Saunders S.J. Barrangou R. Brouns S.J.J. Charpentier E. Haft D.H. et al.An updated evolutionary classification of CRISPR-Cas systems.Nat. Rev. Microbiol. 2015; 13: 722-736Crossref PubMed Scopus (46) Google Scholar). Although Cas1 and Cas2 may be the minimal proteins required for spacer acquisition in some systems, the association of Cas1, Cas2, and the interference machinery allows the host to coordinate robust adaptive immunity in Type I systems. The mechanism underlying the preference for foreign over self DNA during protospacer selection remained poorly understood until a recent study on spacer acquisition during naive acquisition. Spacer acquisition in E. coli was shown to be highly dependent on DNA replication, and foreign-derived spacers were preferred over self-derived spacers by about 100- to 1,000-fold (Levy et al., 2015Levy A. Goren M.G. Yosef I. Auster O. Manor M. Amitai G. Edgar R. Qimron U. Sorek R. CRISPR adaptation biases explain preference for acquisition of foreign DNA.Nature. 2015; 520: 505-510Crossref PubMed Scopus (28) Google Scholar). Analysis of the source of self-derived spacers demonstrated that protospacers were acquired largely from genomic loci predicted to frequently generate stalled replication forks and double-stranded DNA breaks (Levy et al., 2015Levy A. Goren M.G. Yosef I. Auster O. Manor M. Amitai G. Edgar R. Qimron U. Sorek R. CRISPR adaptation biases explain preference for acquisition of foreign DNA.Nature. 2015; 520: 505-510Crossref PubMed Scopus (28) Google Scholar). Such harmful dsDNA breaks are repaired by the helicase/nuclease RecBCD complex, which degrades the broken ends until reaching a Chi-site, after which only the 5′ end is degraded (Dillingham and Kowalczykowski, 2008Dillingham M.S. Kowalczykowski S.C. RecBCD enzyme and the repair of double-stranded DNA breaks.Microbiol. Mol. Biol. Rev. 2008; 72: 642-671Crossref PubMed Scopus (190) Google Scholar). Due to the lower frequency of Chi sites in foreign DNA, RecBCD is predicted to preferentially degrade plasmids and viral DNA, resulting in the generation of candidate protospacer substrates for Cas1 and Cas2 (Levy et al., 2015Levy A. Goren M.G. Yosef I. Auster O. Manor M. Amitai G. Edgar R. Qimron U. Sorek R. CRISPR adaptation biases explain preference for acquisition of foreign DNA.Nature. 2015; 520: 505-510Crossref PubMed Scopus (28) Google Scholar) (Figure 2A). RecBCD degrades DNA asymmetrically, yielding single-stranded fragments ranging from tens to hundreds of nucleotides long from one strand and kilobases long from the other (Dillingham and Kowalczykowski, 2008Dillingham M.S. Kowalczykowski S.C. RecBCD enzyme and the repair of double-stranded DNA breaks.Microbiol. Mol. Biol. Rev. 2008; 72: 642-671Crossref PubMed Scopus (19
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