Abstract
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Recent advances with the RNA-mediated Cas9 endonuclease derived from clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems have dramatically transformed our ability to specifically modify intact genomes of diverse cells and organisms. The CRISPR–Cas system has been adapted as an efficient, facile, and robust gene-targeting technology with the potential for high-throughput and multiplexed genome engineering. Exciting breakthroughs in understanding the mechanisms of the CRISPR–Cas system and its enormous potential for applications across basic science, agricultural and biotechnology.
Similar content being viewed by others
References
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819):1709–1712
Beloglazova N, Petit P, Flick R, Brown G, Savchenko A, Yakunin AF (2011) Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference. EMBO J 30(22):4616–4627
Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A, Heimerikx M, Kerkhoven RM, Madiredjo M, Nijkamp W, Weigelt B (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428(6981):431–437
Bhaya D, Davison M, Barrangou R (2011) CRISPR–Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 45:273–297. doi:10.1146/annurev-genet-110410-132430
Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA (2013) Programmable repression and activation of bacterial gene expression using an engineered CRISPR–Cas system. Nucleic Acids Res 41(15):7429–7437. doi:10.1093/nar/gkt520
Bogdanove AJ, Voytas DF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333(6051):1843–1846
Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (2005) Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151(8):2551–2561
Boutros M, Kiger AA, Armknecht S, Kerr K, Hild M, Koch B, Haas SA, Paro R, Perrimon N (2004) Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303(5659):832–835
Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, Van Der Oost J (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321(5891):960–964
Cady KC, Bondy-Denomy J, Heussler GE, Davidson AR, O’Toole GA (2012) The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages. J Bacteriol 194(21):5728–5738
Carroll D (2012) A CRISPR approach to gene targeting. Mol Ther 20(9):1658–1660
Carroll D (2013) Staying on target with CRISPR–Cas. Nat Biotechnol 31(9):807–809
Carte J, Wang R, Li H, Terns RM, Terns MP (2008) Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev 22(24):3489–3496
Carte J, Pfister NT, Compton MM, Terns RM, Terns MP (2010) Binding and cleavage of CRISPR RNA by Cas6. RNA 16(11):2181–2188
Chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ (2013) Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res 23(4):465–472. doi:10.1038/cr.2013.45
Charpentier E, Doudna JA (2013) Biotechnology: rewriting a genome. Nature 495(7439):50–51
Cho SW, Kim S, Kim JM, Kim J-S (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232
Chylinski K, Le Rhun A, Charpentier E (2013) The tracrRNA and Cas9 families of type II CRISPR–Cas immunity systems. RNA Biol 10(5):726–737. doi:10.4161/rna.24321
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823
Datsenko KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E (2012) Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat Commun 3:945
Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471(7340):602–607. doi:10.1038/nature09886
Deveau H, Barrangou R, Garneau JE, Labonte J, Fremaux C, Boyaval P, Romero DA, Horvath P, Moineau S (2008) Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 190(4):1390–1400. doi:10.1128/JB.01412-07
Deveau H, Garneau JE, Moineau S (2010) CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol 64:475–493
DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR–Cas systems. Nucleic Acids Res 41(7):4336–4343. doi:10.1093/nar/gkt135
Dickinson DJ, Ward JD, Reiner DJ, Goldstein B (2013) Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods 10(10):1028–1034
Echeverri CJ, Beachy PA, Baum B, Boutros M, Buchholz F, Chanda SK, Downward J, Ellenberg J, Fraser AG, Hacohen N (2006) Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat Methods 3(10):777–779
Feng Z, Zhang B, Ding W, Liu X, Yang DL, Wei P, Cao F, Zhu S, Zhang F, Mao Y, Zhu JK (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 23(10):1229–1232
Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang DL, Wang Z, Zhang Z, Zheng R, Yang L, Zeng L, Liu X, Zhu JK (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA. doi:10.1073/pnas.1400822111
Fonfara I, Le Rhun A, Chylinski K, Makarova KS, Lecrivain AL, Bzdrenga J, Koonin EV, Charpentier E (2013) Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR–Cas systems. Nucleic Acids Res. doi:10.1093/nar/gkt1074
Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA (2013) Heritable genome editing in C. elegans via a CRISPR–Cas9 system. Nat Methods 10(8):741–743
Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells. Nat Biotechnol 31(9):822–826. doi:10.1038/nbt.2623
Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31(7):397–405. doi:10.1016/j.tibtech.2013.04.004
Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadan AH, Moineau S (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468(7320):67–71. doi:10.1038/nature09523
Garside EL, Schellenberg MJ, Gesner EM, Bonanno JB, Sauder JM, Burley SK, Almo SC, Mehta G, MacMillan AM (2012) Cas5d processes pre-crRNA and is a member of a larger family of CRISPR RNA endonucleases. RNA 18(11):2020–2028
Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci 109(39):E2579–E2586
Gesner EM, Schellenberg MJ, Garside EL, George MM, MacMillan AM (2011) Recognition and maturation of effector RNAs in a CRISPR interference pathway. Nat Struct Mol Biol 18(6):688–692
Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154(2):442–451. doi:10.1016/j.cell.2013.06.044
Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP (2009) RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139(5):945–956. doi:10.1016/j.cell.2009.07.040
Hannon GJ (2002) RNA interference. Nature 418(6894):244–251
Hatoum-Aslan A, Maniv I, Marraffini LA (2011) Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc Natl Acad Sci 108(52):21218–21222
Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA (2010) Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329(5997):1355–1358. doi:10.1126/science.1192272
Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29(8):731–734
Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327(5962):167–170
Hruscha A, Krawitz P, Rechenberg A, Heinrich V, Hecht J, Haass C, Schmid B (2013) Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development 140(24):4982–4987
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31(9):827–832. doi:10.1038/nbt.2647
Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JJ, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR–Cas system. Nat Biotechnol 31(3):227–229
Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169(12):5429–5433
Jao L-E, Wente SR, Chen W (2013) Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci 110(34):13904–13909
Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013a) RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nat Biotechnol 31(3):233–239. doi:10.1038/nbt.2508
Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013b) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41(20):e188. doi:10.1093/nar/gkt780
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821
Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. Elife 2:e00471
Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, Anders C, Hauer M, Zhou K, Lin S, Kaplan M, Iavarone AT, Charpentier E, Nogales E, Doudna JA (2014) Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science (New York, NY)
Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul Ü, Wurm R, Wagner R (2011) Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol 18(5):529–536
Joung JK, Sander JD (2012) TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14(1):49–55
Kaelin WG (2012) Use and abuse of RNAi to study mammalian gene function. Science 337(6093):421–422
Klug A (2010) The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu Rev Biochem 79:213–231
Koonin EV, Makarova KS (2009) CRISPR–Cas: an adaptive immunity system in prokaryotes. F1000 biology reports 1:95
Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS (2013) CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc 8(11):2180–2196. doi:10.1038/nprot.2013.132
Li T, Liu B, Spalding MH, Weeks DP, Yang B (2012) High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat Biotechnol 30(5):390–392
Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu X (2013a) Heritable gene targeting in the mouse and rat using a CRISPR–Cas system. Nat Biotechnol 31(8):681–683
Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J (2013b) Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31:688–691. doi:10.1038/nbt.265010.1038/nbt.2623
Li W, Teng F, Li T, Zhou Q (2013c) Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR–Cas systems. Nat Biotechnol 31(8):684–686
Lopez-Sanchez MJ, Sauvage E, Da Cunha V, Clermont D, Ratsima Hariniaina E, Gonzalez-Zorn B, Poyart C, Rosinski-Chupin I, Glaser P (2012) The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome. Mol Microbiol 85(6):1057–1071
Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV (2006) 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 1(1):7
Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF (2011) Evolution and classification of the CRISPR–Cas systems. Nat Rev Microbiol 9(6):467–477. doi:10.1038/nrmicro2577
Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM (2013a) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31(9):833–838. doi:10.1038/nbt.2675
Mali P, Esvelt KM, Church GM (2013b) Cas9 as a versatile tool for engineering biology. Nat Methods 10(10):957–963. doi:10.1038/nmeth.2649
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013c) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826
Marraffini LA, Sontheimer EJ (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322(5909):1843–1845
Marraffini LA, Sontheimer EJ (2010a) CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 11(3):181–190. doi:10.1038/nrg2749
Marraffini LA, Sontheimer EJ (2010b) Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463(7280):568–571
Marx J (2007) New bacterial defense against phage invaders identified. Science 315(5819):1650–1651
Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu LJ (2013) Targeted mutagenesis in rice using CRISPR–Cas system. Cell Res 23(10):1233–1236. doi:10.1038/cr.2013.123
Mojica FJ, García-Martínez J, Soria E (2005) Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 60(2):174–182
Moscou MJ, Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326(5959):1501
Mulepati S, Bailey S (2011) Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3). J Biol Chem 286(36):31896–31903
Mussolino C, Cathomen T (2013) RNA guides genome engineering. Nat Biotechnol 31(3):208–209
Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31(8):691–693
Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31(9):839–843. doi:10.1038/nbt.2673
Pennisi E (2013) The CRISPR craze. Science 341:833–836
Pourcel C, Salvignol G, Vergnaud G (2005) CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151(3):653–663
Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5):1173–1183. doi:10.1016/j.cell.2013.02.022
Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F (2013a) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6):1380–1389. doi:10.1016/j.cell.2013.08.021
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013b) Genome engineering using the CRISPR–Cas9 system. Nat Protoc 8(11):2281–2308. doi:10.1038/nprot.2013.143
Rusk N (2014) CRISPRs and epigenome editing. Nat Methods 11(1):28
Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V (2011) The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res 39(21):9275–9282
Sashital DG, Jinek M, Doudna JA (2011) An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3. Nat Struct Mol Biol 18(6):680–687
Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2013) Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 303:84–87
Shan Q, Wang Y, Chen K, Liang Z, Li J, Zhang Y, Zhang K, Liu J, Voytas DF, Zheng X (2013a) Rapid and efficient gene modification in rice and Brachypodium using TALENs. Molecular plant 6(4):1365–1368
Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu J-L (2013b) Targeted genome modification of crop plants using a CRISPR–Cas system. Nat Biotechnol 31(8):686–688
Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X (2013) Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res 23:720–723
Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V (2011) Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J 30(7):1335–1342
Sontheimer EJ, Marraffini LA (2010) Microbiology: slicer for DNA. Nature 468(7320):45–46
Sorek R, Kunin V, Hugenholtz P (2008) CRISPR—a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol 6(3):181–186
Streubel J, Blücher C, Landgraf A, Boch J (2012) TAL effector RVD specificities and efficiencies. Nat Biotechnol 30(7):593–595
Swarts DC, Mosterd C, van Passel MW, Brouns SJ (2012) CRISPR interference directs strand specific spacer acquisition. PLoS One 7(4):e35888
Terns MP, Terns RM (2011) CRISPR-based adaptive immune systems. Curr Opin Microbiol 14(3):321–327
van der Oost J, Jore MM, Westra ER, Lundgren M, Brouns SJ (2009) CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem Sci 34(8):401–407
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013a) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918. doi:10.1016/j.cell.2013.04.025
Wang T, Wei JJ, Sabatini DM, Lander ES (2013b) Genetic screens in human cells using the CRISPR/Cas9 system. Science 303:80–84
Waters LS, Storz G (2009) Regulatory RNAs in bacteria. Cell 136(4):615–628
Weinthal D, Tovkach A, Zeevi V, Tzfira T (2010) Genome editing in plant cells by zinc finger nucleases. Trends Plant Sci 15(6):308–321
Wiedenheft B, Lander GC, Zhou K, Jore MM, Brouns SJ, van der Oost J, Doudna JA, Nogales E (2011a) Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477(7365):486–489
Wiedenheft B, van Duijn E, Bultema JB, Waghmare SP, Zhou K, Barendregt A, Westphal W, Heck AJ, Boekema EJ, Dickman MJ (2011b) RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci 108(25):10092–10097
Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482(7385):331–338. doi:10.1038/nature10886
Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J (2013) Correction of a genetic disease in mouse via use of CRISPR–Cas9. Cell Stem Cell 13(6):659–662. doi:10.1016/j.stem.2013.10.016
Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154(6):1370–1379. doi:10.1016/j.cell.2013.08.022
Yosef I, Goren MG, Qimron U (2012) Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res 40(12):5569–5576
Yu Z, Ren M, Wang Z, Zhang B, Rong YS, Jiao R, Gao G (2013) Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics 195(1):289–291
Zhang F, Maeder ML, Unger-Wallace E, Hoshaw JP, Reyon D, Christian M, Li X, Pierick CJ, Dobbs D, Peterson T (2010) High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proc Natl Acad Sci 107(26):12028–12033
Zhang Y, Heidrich N, Ampattu BJ, Gunderson CW, Seifert HS, Schoen C, Vogel J, Sontheimer EJ (2013a) Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol Cell 50(4):488–503
Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ, Voytas DF (2013b) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol 161(1):20–27
Acknowledgments
We thank anonymous reviewers and the journal editor for their critical comments on the manuscript. We apologise to colleagues for not being able to cite all relevant and earlier papers because of space limitations and the focus of the article on recent research.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Liu, L., Fan, XD. CRISPR–Cas system: a powerful tool for genome engineering. Plant Mol Biol 85, 209–218 (2014). https://doi.org/10.1007/s11103-014-0188-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11103-014-0188-7