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Reviews and Monographs Progress in Biochemistry and Biophysics 2013, 40(8): 691~702 wwwpibbaccn * CRISPR/Cas9 ( 100193) Clustered regularly interspaced short palindromic repeats

1 Reviews and Monographs Progress in Biochemistry and Biophysics 2013, 40(8): 691~702 wwwpibbaccn * CRISPR/Cas9 ( ) Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 (zinc finger endonuclease ZFN) (transcription activator-like effector nuclease TALEN) InDel CRISPR/Cas CRISPR CRISPR/Cas Q7 DOI: /SPJ (transcription activator-like [4 6] effector TALE) DNA 2010 TALEN [7] (engineered endonuclease EEN) (zinc finger endonuclease ZFN) 2012 TALEN (Science) [9] DNA ZFN TALEN ZFN DNA [1] Fok Fok 2013 DNA clustered regularly ZFN interspaced short palindromic repeats (CRISPR)/ CRISPR-associated(Cas)9 ips [2] [10] HIV CRISPR ZFN( CCR ) CRISPR/Cas [3] ZFN CRISPR/Cas9 (transcription activator-like effector nuclease TALEN) ZFN2009 (Xanthamonas) [8] TALEN ZFN * (CARS-37) ** Tel: , qingyongmeng@gmailcom

2 692 Prog Biochem Biophys 2013; 40 (8) 1 CRISPR/Cas 5' tracrrna Cas Cas9 Cas1 Cas2 CRISPR/Cas ( 1) Csn2 3' CRISPR (Streptococcus pyogenes SF370) (spacers) (direct repeats) Type CRISPR/Cas tracrrna Cas9 Cas1 Cas2Csn2 CRISPR 5' 3' tracrrna Pre-crRNA Cas9 RNase DNA PAM Fig 1 Schematic of CRISPR/Cas9 mediated DNA double-strand break 1 CRISPR/Cas9 TracrRNA RNase Cas9 pre-rna TracrRNA crrna Cas9 2 CRISPR CRISPR K12 CRISPR (spacer) [11] [15 17] clustered regularly interspaced short palindromic CRISPR RNAi repeats(crispr) [12 14]

3 2013; 40 (8), CRISPR/Cas9 693 [18] [26, 28 29] 2007 Barrangou [19] Cas CRSPR 2008 Cas [30] Marraffini [20] CRISPR CRISPR Cas Cas CRISPR CRISPR/Cas CRISPR 3 CRISPR Cas1 Cas2 CRISPR RNA CRISPRdb CRISPI CRISPR CRISPR/Cas Type Type CRISPRdb Type CRSPR (http Type CRISPR/Cas Cas //crisprupsudfr) [21 22] CRSPI Cas 6 CRISPRs CPRISPRdb (http Cas3 //crispigeneouestorg/) CRISPR [31] Cas crrna 90% 40% CRISPR CASCAD(CRISPR CRISPR associated complex for antivirus defense) CASCAD [23] CRISPR DNA CASCAD 21~72 bp crrna DNA R CRISPR Cas3 R Cas3 [32 34] CRISPR (Haliangium ochraceum DSM 14365) 587 Type [24] CRISPR 21 ~ Cas9 ( ) crrna 48 bp DNA CRISPR [35] Cas9 N 5' 3' GTTT/g Ruc GAAAC HNH RNA Type CRISPR/Cas CRISPR/Cas Cas tracrrna(trans-activating crrna) [14, 24 26] CRISPR RNase Cas9 crrna tracrrna crrna Cas RNA Cas9 [27] Cas DNA CRISPR RNA [36] CRISPR/Cas Type Cas10 Cas CRISPR RNA Type CASCAD CRISPR [37] Cas10 crrna DNA Type Type A CRISPR CRISPR Type B CRISPR/Cas Cas Type A mrna [38] Cas CRISPR/Cas Type B

4 694 Prog Biochem Biophys 2013; 40 (8) Type CRISPR/Cas CRISPR (spacer) DNA [20] CRISPR/Cas DNA DNA CRISPR/Cas CRRSPR 5' Type (repeats) CRISPR Type 5' 3' Type [39] protospacer protospacer 5' 3' PAM CRISPR/Cas (protospacer adjacent motifs) 2~5 protospacer 1~4 [41] CRISPR/Cas DNA PAM [24, 28 29, 40] protospacer CRISPR 5' 4 CRISPR/Cas ( 2) CRISPR/Cas Cas1 Cas2 CRISPR [19, 42] Type CRISRP/ Cas CRIPSR ( Csn2 ) CRISPR/Cas [19, 43] PAM 5' 3' CAC GACCCC GAGG AGAGGG AACGC AGGG AGAC TGAGG TG GTGC TGGGG CTCC TCTCCCTTGC GTCCCT CTGA C TCCAC 3' 5' Cas CRISPR Fig 2 New spcacer of CRISPR acqusition 2 CRISPR CRISPR crrna Cas9 RNase [36] CRISPR RNA(pre-crRNA) Cas tracrrna CRISPR [44, 49] RNA RNA crrna CRISPR [43 48] Type CRISPR/Cas [45 46, 48, 50 51]

5 2013; 40 (8), CRISPR/Cas9 695 CRISPR/Cas 6 CRISRP/Cas crrna Cas CRISPR/Cas DNA DNA crrna CRISPR/Cas DNA CRISPR crrna (spacer) DNA [55] [19, 52] CRISPR/Cas spacer protospacer [34 35, 53] crrna Cas 5 CRISPR PAM protospacer CRISPR/Cas CRISPR/Cas (engineered endonuclease EEN) crrna DNA crrna CRISPR/Cas Type pre-crrna crrna tracrrna Cas9 (Streptococcus CRISPR/Cas pyogenes SF370) Type CRISPR [56 58] crrna ENN Martin Type spacer 5' 3' CRISPR/Cas9 repeat DNA crrna [59 60] tracrrna DNA (protopacer) tracrrna 5' crrna 3' ( 13 bp) protospacer crrna crrna protospacer tracrrna crrna repeat protospacer tracrrna crrna DNA RNA (guide RNA grna) grna tracrrna crrna ( 3) [54] CRISPR/Cas Cas9 N RuvC CRRISP/Cas HNH CRISPR spacer RuvC HNH Xop411 Cas9 Xop411 PAM 5' protospacer PAM (proto-spacer adjacent motifs) DNA protospacer PAM 3~8 PAM CRISPR/Cas 2013 MIT Zhang [53] DNA CRISPR CRISPR/Cas9 293T EMX1 (spacer) PAM PVALB Nero2A Th CRISPR DNA DNA [58] crrna RNase

696 Prog Biochem Biophys 2013; 40 (8) CMV NLS-Cas9-NLS PolyA U6 RNA RNA Cas9 RNA RNA Cas9: grna Fig 3 Schematic of grna (chimeric RNA) guide Cas9 site-specific cleavage in animal genome 3 grna

6 696 Prog Biochem Biophys 2013; 40 (8) CMV NLS-Cas9-NLS PolyA U6 RNA RNA Cas9 RNA RNA Cas9: grna Fig 3 Schematic of grna (chimeric RNA) guide Cas9 site-specific cleavage in animal genome 3 grna (chemricrna) Cas9 (Science) Mali [57] ZFN TALEN [62 64] CRISPR/Cas9 CRISPR/Cas9 293T K652 CRISPR/Cas9 DNA ( CRISPR/Cas9 ) crrna:tracrrna grna Zhang Zhang grna grna crrna tracrrna grna Mali crrna grna tracrrna grna Cas9 Cas9 Nature Biotechnology Cell Research N C Mali grna Zhang Cas9 [56, 61] [58] Mali grna 3' NLS Cas9 tracrrna crrna Mali Cas9 C tracrrna 3' grna Cas9 CRSIPR/Cas9 [57] Shen [65] Cas9 N N C

7 2013; 40 (8), CRISPR/Cas9 697 Cas9 N Cas9 32 Cas9 NLS (<10-7 ) ZFN TALEN CRSPR/Cas9 Cas9 C Cas9 ZFN TALEN [71 74] 61 CRISPR/Cas9 CRISPR/Cas9 ZFN TALEN (single nucleotide CRISPR/Cas9 polymorphism SNP) CRISRP/Cas9 DNA IFG2 2 SNP IGF2 [75] (NHEJ) (HR) MSTN 3' SNP MSTN [57] [76] NHEJ Cas9 SNP SNP NHEJ Ding [66] SNP Cell Stem Cell ips CRISPR/Cas9 TALEN 2 SNP ips [67 70] ips ZFN TALEN CRISPR/ Cas9 ips CRISPR/ Cas9 CRISPR/Cas9 ips CRISPR/Cas9 8 bp TALEN ZNF 1/125 bp [77] TALEN CRISPR/Cas9 ZFN 500 bp CRISPR/Cas9 ips CRISPR/Cas9 CRISPR/Cas9 62 CRISPR/Cas9 ZFN TALEN 1997 Cas9 DNA Cas9 Cas9 ZFN TALEN CRISPR/Cas9 100% DNA DNA

8 698 Prog Biochem Biophys 2013; 40 (8) ZFN TALEN ( ) TALEN DNA RVD ZFN TALEN DNA 10bp ZFN ZFN TALEN TALEN CRISPR/Cas9 PAM (NGG) RVD PAM 11 bp 14 bp (PAM+ ) CRISPR/Cas9 CRISPR/Cas9 14 bp DNA 20~30 bp CRISPR/Cas9 ZFN TALEN CRISPR/Cas9 ( ) [78] SNP ZFN TALEN CRISPR/Cas9 NHEJ HR ZNF TALEN CRISPR/Cas9 ZFN TALEN ZFN DNA PCR TA ZFN TALEN ( Cel T7E1 ) DNA 8 ( VP16 KRAB) [79 81] Qi [82] CRISPR 25 CRIPSR/Cas9 CRISPR CRISPR Cas9 DNA grna CRISPR RNA [83] CRISPR [84] CRISPR [85] 100% Cas9 VP16 CRISPR/Cas Cas9 CRISPR ZFN TALEN CRISPR CRISPR spacer spacer CRISPR 5' 7 CRISPR/Cas9 CRISPR/Cas9 Type CRISPR ZFN TALEN ENN ZFN TALEN ZFN TALEN DNA ZFN

9 2013; 40 (8), CRISPR/Cas9 699 ENN 326(5959): ENN CRISPR/Cas9 3' PAM (NGG) in eukaryotes Nucleic acids Res, 2011, 39(14): TALEN ZFN CRISPR/Cas bp (PAM 3' 11 bp) 338(6114): CRISPR/Cas9 ZFN TALEN [66, 78] CRISPR/Cas9 ZFN TALEN Cas9 CRISPR/Cas9 ZFN TALEN? Journal of Integrative Biology, 2002, 6(1): CRISPR/Cas9 replicon partitioning Mol Microbiol, 1995, 17(1): Cas9 CRISPR/Cas9 ZFN TALEN a b ZFN TALEN ccrispr/cas9 ZFN TZLEN foreign genetic elements J Mol Evol, 2005, 60(2): CRISPR/Cas9 [1] Kim Y G, Cha J, Chandrasegaran S Hybrid restriction enzymes: zinc finger fusions to Fok cleavage domain Proc Natl Acad Sci USA, 1996, 93(3): [2] Urnov F D, Rebar E J, Holmes M C, et al Genome editing with engineered zinc finger nucleases Nat Rev Genet, 2010, 11 (9): [3] Perez E E, Wang J, Miller J C, et al Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases Nat Biotechnol, 2008, 26(7): [4] Moscou M J, Bogdanove A J A simple cipher governs DNA recognition by TAL effectors Science, 2009, 326(5959): 1501 [5] Boch J, Scholze H, Schornack S, et al Breaking the code of DNA binding specificity of TAL-type effectors Science, 2009, [6] Boch J, Bonas U Xanthomonas AvrBs3 family-type effectors: discovery and function Annu Rev Phytopathol, 2010, 48: [7] Li T, Huang S, Zhao X, et al Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement [8] Joung J K, Sander J D TALENs: a widely applicable technology for targeted genome editing Nat Rev Mol Cell Biol, 2013, 14 (1): [9] Breakthrough of the year The runners-up Science, 2012, [10] Mussolino C, Cathomen T RNA guides genome engineering Nat Biotechnol, 2013, 31(3): [11] Ishino Y, Shinagawa H, Makino K, et al 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(12): [12] Jansen R, van Embden J D, Gaastra W, et al Identification of a novel family of sequence repeats among prokaryotes Omics: a [13] Mojica F J, Ferrer C, Juez G, et al Long stretches of short tandem repeats are present in the largest replicons of the archaea Haloferax mediterranei and Haloferax volcanii and could be involved in [14] Jansen R, Embden J D, Gaastra W, et al Identification of genes that are associated with DNA repeats in prokaryotes Mol Microbiol, 2002, 43(6): [15] Bolotin A, Quinquis B, Sorokin A, et al Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin Microbiology, 2005, 151 (Pt 8): [16] Mojica F J, Diez-Villasenor C, Garcia-Martinez J, et al Intervening sequences of regularly spaced prokaryotic repeats derive from [17] Pourcel 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(Pt 3): [18] Makarova K S, Grishin N V, Shabalina S A, et al 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 Biol Direct, 2006, 1: 7 [19] Barrangou R, Fremaux C, Deveau H, et al CRISPR provides acquired resistance against viruses in prokaryotes Science, 2007, 315(5819): [20] Marraffini L A, Sontheimer E J CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA Science, 2008, 322(5909): [21] Grissa I, Vergnaud G, Pourcel C CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats Nucl Acids Res, 2007, 35(Web Server issue): W52 57

10 700 Prog Biochem Biophys 2013; 40 (8) [22] Rousseau C, Gonnet M, Le Romancer M, et al CRISPI: a CRISPR interactive database Bioinformatics, 2009, 25(24): [23] Grissa I, Vergnaud G, Pourcel C The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats BMC Bioinformatics, 2007, 8: 172 [24] Godde J S, Bickerton A The repetitive DNA elements called CRISPRs and their associated genes: evidence of horizontal transfer among prokaryotes J Mol Evol, 2006, 62(6): [25] Deveau H, Garneau JE, Moineau S CRISPR/Cas system and its role in phage-bacteria interactions Annu Rev Microbiol, 2010, 64: [26] Kunin V, Sorek R, Hugenholtz P Evolutionary conservation of sequence and secondary structures in CRISPR repeats Genome Biol, 2007, 8(4): R61 [27] Makarova K S, Aravind L, Grishin N V, et al A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis Nucleic Acids Res, 2002, 30(2): [28] Heidelberg J F, Nelson W C, Schoenfeld T, et al Germ warfare in a microbial mat community: CRISPRs provide insights into the co-evolution of host and viral genomes PloS One, 2009, 4 (1): e4169 [29] Horvath P, Coute-Monvoisin A C, Romero D A, et al Comparative analysis of CRISPR loci in lactic acid bacteria genomes Int J Food Microbiol, 2009, 131(1): [30] Haft D H, Selengut J, Mongodin E F, et al A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes PLOS Comput Biol, 2005, 1(6): e60 [31] Sinkunas T, Gasiunas G, Fremaux C, et al Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system EMBO J, 2011, 30(7): [32] Haurwitz R E, Jinek M, Wiedenheft B, et al Sequence- and structure-specific RNA processing by a CRISPR endonuclease Science, 2010, 329(5997): [33] Wiedenheft B, Zhou K, Jinek M, et al Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense Structure, 2009, 17(6): [34] Wiedenheft B, van Duijn E, Bultema J B, et al RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions Proc Natl Acad Sci USA, 2011, 108(25): [35] Garneau J E, Dupuis M E, Villion M, et al The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA Nature, 2010, 468(7320): [36] Deltcheva E, Chylinski K, Sharma C M, et al CRISPR RNA maturation by trans-encoded small RNA and host factor RNase Nature, 2011, 471(7340): [37] Anantharaman V, Iyer L M, Aravind L Presence of a classical RRM-fold palm domain in Thg1-type 3'- 5'nucleic acid polymerases and the origin of the GGDEF and CRISPR polymerase domains Biol Direct, 2010, 5: 43 [38] Hale C R, Zhao P, Olson S, et al RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex Cell, 2009, 139(5): [39] Terns M P, Terns R M CRISPR-based adaptive immune systems Curr Opin Microbiol, 2011, 14(3): [40] Portillo M C, Gonzalez J M CRISPR elements in the Thermococcales: evidence for associated horizontal gene transfer in Pyrococcus furiosus J Appl Genet, 2009, 50(4): [41] Mojica F J, Diez-Villasenor C, Garcia-Martinez J, et al Short motif sequences determine the targets of the prokaryotic CRISPR defence system Microbiol, 2009, 155(Pt 3): [42] Beloglazova N, Brown G, Zimmerman M D, et al A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats J Biol Chem, 2008, 283(29): [43] Brouns S J, Jore M M, Lundgren M, et al Small CRISPR RNAs guide antiviral defense in prokaryotes Science, 2008, 321 (5891): [44] Pougach K, Semenova E, Bogdanova E, et al Transcription, processing and function of CRISPR cassettes in Escherichia coli Mol Microbiol, 2010, 77(6): [45] Pul U, Wurm R, Arslan Z, et al Identification and characterization of E coli CRISPR-cas promoters and their silencing by H-NS Mol Microbiol, 2010, 75(6): [46] Agari Y, Sakamoto K, Tamakoshi M, et al Transcription profile of Thermus thermophilus CRISPR systems after phage infection J Mol Biol, 2010, 395(2): [47] Hale C, Kleppe K, Terns R M, et al Prokaryotic silencing (psi) RNAs in Pyrococcus furiosus Rna, 2008, 14(12): [48] Lillestol R K, Shah S A, Brugger K, et al CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties Molecular Microbiology, 2009, 72(1): [49] Phok K, Moisan A, Rinaldi D, et al Identification of CRISPR and riboswitch related RNAs among novel noncoding RNAs of the euryarchaeon Pyrococcus abyssi BMC Genomics, 2011, 12: 312 [50] Lillestol R K, Redder P, Garrett R A, et al A putative viral defence mechanism in archaeal cells Archaea, 2006, 2(1): [51] Shinkai A, Kira S, Nakagawa N, et al Transcription activation mediated by a cyclic AMP receptor protein from Thermus thermophilus HB8 J Bacteriol, 2007, 189(10): [52] Deveau H, Barrangou R, Garneau J E, et al Phage response to CRISPR-encoded resistance in Streptococcus thermophilus J Bacteriol, 2008, 190(4): [53] Semenova E, Nagornykh M, Pyatnitskiy M, et al Analysis of CRISPR system function in plant pathogen Xanthomonas oryzae FEMS Microbiology Letters, 2009, 296(1): [54] Marraffini L A, Sontheimer E J Self versus non-self discrimination during CRISPR RNA-directed immunity Nature, 2010, 463(7280): [55] Barrangou R, Horvath P CRISPR: new horizons in phage resistance and strain identification Annu Rev Food Sci Technol, 2012, 3: [56] Hwang W Y, Fu Y, Reyon D, et al Efficient genome editing in zebrafish using a CRISPR-Cas system Nat Biotechnol, 2013, 31(3):

11 2013; 40 (8), CRISPR/Cas [57] Mali P, Yang L, Esvelt K M, et al RNA-guided human genome engineering via Cas9 Science, 2013, 339(6121): [58] Cong L, Ran F A, Cox D, et al Multiplex genome engineering using CRISPR/Cas systems Science, 2013, 339(6121): [59] Jinek M, Chylinski K, Fonfara I, et al A programmable dual-rna-guided DNA endonuclease in adaptive bacterial immunity Science, 2012, 337(6096): [60] Gasiunas G, Barrangou R, Horvath P, et al Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria Proc Natl Acad Sci USA, 2012, 109(39): E [61] Chang N, Sun C, Gao L, et al Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos Cell Res, 2013, 23(4): [62] Kim Y, Kweon J, Kim A, et al A library of TAL effector nucleases spanning the human genome Nat Biotechnol, 2013, 31(3): [63] Bultmann S, Morbitzer R, Schmidt C S, et al Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers Nucleic Acids Res, 2012, 40(12): [64] Chen S, Oikonomou G, Chiu C N, et al A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly Nucleic Acids Res, 2013, 41(4): [65] Shen B, Zhang J, Wu H, et al Generation of gene-modified mice via Cas9/RNA-mediated gene targeting Cell Res, 2013, 23 (5): [66] Ding Q, Regan S N, Xia Y, et al Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs Cell Stem Cell, 2013, 12(4): [67] Liu G H, Suzuki K, Qu J, et al Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific ipscs Cell Stem Cell, 2011, 8(6): [68] Wang Y, Zheng C G, Jiang Y, et al Genetic correction of beta-thalassemia patient-specific ips cells and its use in improving hemoglobin production in irradiated SCID mice Cell Res, 2012, 22(4): [69] Kazuki Y, Hiratsuka M, Takiguchi M, et al Complete genetic correction of ips cells from Duchenne muscular dystrophy Molecular therapy: the journal of the American Society of Gene Therapy, 2010, 18(2): [70] Sun N, Longaker M T, Wu J C Human ips cell-based therapy: considerations before clinical applications Cell Cycle, 2010, 9(5): [71] Carlson D F, Tan W, Lillico S G, et al Efficient TALEN-mediated gene knockout in livestock Proc Natl Acad Sci USA, 2012, 109(43): [72] Yu S, Luo J, Song Z, et al Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle Cell Res, 2011, 21(11): [73] Yang D, Yang H, Li W, et al Generation of PPARgamma mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning Cell Res, 2011, 21(6): [74] Hauschild J, Petersen B, Santiago Y, et al Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases Proc Natl Acad Sci USA, 2011, 108(29): [75] Van Laere A S, Nguyen M, Braunschweig M, et al A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig Nature, 2003, 425(6960): [76] Clop A, Marcq F, Takeda H, et al A mutation creating a potential illegitimate microrna target site in the myostatin gene affects muscularity in sheep Nat Genet, 2006, 38(7): [77] Tan W S, Carlson D F, Walton M W, et al Precision editing of large animal genomes Adv Genet, 2012, 80: [78] Wang H, Yang H, Shivalila C S, et al One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering Cell, 2013 [79] Miller J C, Tan S, Qiao G, et al A TALE nuclease architecture for efficient genome editing Nat Biotechnol, 2011, 29(2): [80] Zhang F, Cong L, Lodato S, et al Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription Nat Biotechnol, 2011, 29(2): [81] Morbitzer R, Romer P, Boch J, et al Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors Proc Natl Acad Sci USA, 2010, 107(50): [82] Qi L S, Larson M H, Gilbert L A, et al Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression Cell, 2013, 152(5): [83] Koonin E V, Wolf Y I Is evolution Darwinian or/and Lamarckian? Biol Direct, 2009, 4: 42 [84] Sampson T R, Saroj S D, Llewellyn A C, et al A CRISPR/Cas system mediates bacterial innate immune evasion and virulence Nature, 2013, 497(7448): [85] Horvath P, Barrangou R CRISPR/Cas, the immune system of bacteria and archaea Science, 2010, 327(5962):

12 702 Prog Biochem Biophys 2013; 40 (8) New Method of Genome Editing Derived From CRISPR/Cas9 * FANG Rui, CHANG Fei, Sun Zhao-Lin, LI Ning, MENG Qing-Yong ** (State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing , China) Abstract The type clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 system has been used as a gene-targeting technology as ZFN and TALEN to meditate multiple genome editing Unlike ZFN and TALEN directly binding to the specific DNA sequence to generate a double-strands break, the engineered CRISPR/Cas9 system has been demonstrated that Cas9 nuclease was directed by short RNAs to induce site-specific cleavage in complex genome The advantage of CRPSR/Cas9 system is easy to be constructed and low cost compared with ZFN and TALEN So, it is considered that it will replace existing technique Here we review the CRISPR/Cas development history, classification, mechanism, progress and application of this new genome editing technology This review will provide a useful reference for researchers who are interested in applying this new technique in their studies Key words CRISPR/Cas9, gene targeting, genome editing technology, engineered endonuclease DOI: /SPJ * This work was supported by grants from Earmarked Fund for Modern Agro-industry Technology Research Systems of China (CARAS-37) **Corresponding author Tel: , qingyongmeng@gmailcom Received: May 19, 2013 Accepted: July 29, 2013

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2015 Intellectual Sensitive 30 Physical A T G C DNA DNA 7 24 Zemin Ning Kai Ye BIG BIG 1 104 100101 010-84097548 www.big.ac.cn zhouml@big.ac.cn REV1 FANCD2 DNA 6 18 50 聚焦 70 88 7 22 20 7 3 complex indels