Prokaryotic CRISPR-Cas loci encode proteins that function as an adaptive immune system against infectious viruses and plasmids. Immunity is mediated by Cas nucleases and small RNA guides, which specify a cleavage site within the genome of the invader. In type II CRISPR-Cas systems, the RNA-guided Cas9 nuclease cleaves the DNA. Cas9 can be reprogrammed to create double-strand DNA breaks in the genomes of a variety of organisms, from bacteria to human cells. Repair of Cas9 lesions by homologous recombination or nonhomologous end joining mechanisms can lead to the introduction of specific nucleotide substitutions or indel mutations, respectively. Furthermore, a nuclease-null Cas9 has been developed to regulate endogenous gene expression and to label genomic loci in living cells. Targeted genome editing and gene regulation mediated by Cas9 are easy to program, scale, and multiplex, allowing researchers to decipher the causal link between genetic and phenotypic variation. In this review, we describe the most notable applications of Cas9 in basic biology, translational medicine, synthetic biology, biotechnology, and other fields.


Article metrics loading...

Loading full text...

Full text loading...


Literature Cited

  1. Anton T, Bultmann S, Leonhardt H, Markaki Y. 1.  2014. Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system. Nucleus 5:163–72 [Google Scholar]
  2. Bae S, Park J, Kim JS. 2.  2014. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30:1473–75 [Google Scholar]
  3. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P. 3.  et al. 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–12 [Google Scholar]
  4. Barrangou R, Marraffini LA. 4.  2014. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol. Cell 54:234–44 [Google Scholar]
  5. Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A. 5.  et al. 2004. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428:431–37 [Google Scholar]
  6. Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW. 6.  et al. 2014. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32:1146–50 [Google Scholar]
  7. Bikard D, Hatoum-Aslan A, Mucida D, Marraffini LA. 7.  2012. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. Cell Host Microbe 12:177–86 [Google Scholar]
  8. Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. 8.  2013. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41:7429–37 [Google Scholar]
  9. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S. 9.  et al. 2009. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509–12 [Google Scholar]
  10. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. 10.  2005. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151:2551–61 [Google Scholar]
  11. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ. 11.  et al. 2008. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–64 [Google Scholar]
  12. Capecchi MR. 12.  1989. Altering the genome by homologous recombination. Science 244:1288–92 [Google Scholar]
  13. Carroll D. 13.  2011. Zinc-finger nucleases: a panoramic view. Curr. Gene Ther. 11:2–10 [Google Scholar]
  14. Carte J, Wang R, Li H, Terns RM, Terns MP. 14.  2008. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 22:3489–96 [Google Scholar]
  15. Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W. 15.  et al. 2013. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479–91 [Google Scholar]
  16. Cheng AW, Wang H, Yang H, Shi L, Katz Y. 16.  et al. 2013. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23:1163–71 [Google Scholar]
  17. Cho SW, Kim S, Kim Y, Kweon J, Kim HS. 17.  et al. 2014. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24:132–41 [Google Scholar]
  18. Choulika A, Perrin A, Dujon B, Nicolas JF. 18.  1995. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol. Cell. Biol. 15:1968–73 [Google Scholar]
  19. Citorik RJ, Mimee M, Lu TK. 19.  2014. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat. Biotechnol. 32:1141–45 [Google Scholar]
  20. Cong L, Ran FA, Cox D, Lin S, Barretto R. 20.  et al. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–23 [Google Scholar]
  21. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y. 21.  et al. 2011. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602–7 [Google Scholar]
  22. Deng L, Garrett RA, Shah SA, Peng X, She Q. 22.  2013. A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus. Mol. Microbiol. 87:1088–99 [Google Scholar]
  23. Deveau H, Barrangou R, Garneau JE, Labonte J, Fremaux C. 23.  et al. 2008. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190:1390–400 [Google Scholar]
  24. Deveau H, Garneau JE, Moineau S. 24.  2010. CRISPR/Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbiol. 64:475–93 [Google Scholar]
  25. Dianov GL, Hubscher U. 25.  2013. Mammalian base excision repair: the forgotten archangel. Nucleic Acids Res. 41:3483–90 [Google Scholar]
  26. Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco JA. 26.  2013. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods 10:741–43 [Google Scholar]
  27. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D. 27.  et al. 2013. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31:822–26 [Google Scholar]
  28. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. 28.  2014. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32:279–84 [Google Scholar]
  29. Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R. 29.  et al. 2010. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71 [Google Scholar]
  30. Gasiunas G, Barrangou R, Horvath P, Siksnys V. 30.  2012. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. PNAS 109:E2579–86 [Google Scholar]
  31. Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y. 31.  et al. 2014. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159:647–61 [Google Scholar]
  32. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA. 32.  et al. 2013. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154:442–51 [Google Scholar]
  33. Goldberg GW, Jiang W, Bikard D, Marraffini LA. 33.  2014. Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting. Nature 514:633–37 [Google Scholar]
  34. Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, Beisel CL. 34.  2014. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 5:e00928–13 [Google Scholar]
  35. Gong B, Shin M, Sun J, Jung CH, Bolt EL. 35.  et al. 2014. Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3. PNAS 111:16359–64 [Google Scholar]
  36. Guilinger JP, Thompson DB, Liu DR. 36.  2014. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32:577–82 [Google Scholar]
  37. Hale C, Kleppe K, Terns RM, Terns MP. 37.  2008. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 14:2572–79 [Google Scholar]
  38. Hale CR, Zhao P, Olson S, Duff MO, Graveley BR. 38.  et al. 2009. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139:945–56 [Google Scholar]
  39. Hatoum-Aslan A, Maniv I, Marraffini LA. 39.  2011. Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. PNAS 108:21218–22 [Google Scholar]
  40. Hatoum-Aslan A, Samai P, Maniv I, Jiang W, Marraffini LA. 40.  2013. A ruler protein in a complex for antiviral defense determines the length of small interfering CRISPR RNAs. J. Biol. Chem. 288:27888–97 [Google Scholar]
  41. Heler R, Marraffini LA, Bikard D. 41.  2014. Adapting to new threats: the generation of memory by CRISPR-Cas immune systems. Mol. Microbiol. 93:1–9 [Google Scholar]
  42. Hochstrasser ML, Taylor DW, Bhat P, Guegler CK, Sternberg SH. 42.  et al. 2014. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference. PNAS 111:6618–23 [Google Scholar]
  43. Holkers M, Maggio I, Henriques SF, Janssen JM, Cathomen T, Goncalves MA. 43.  2014. Adenoviral vector DNA for accurate genome editing with engineered nucleases. Nat. Methods 11:1051–57 [Google Scholar]
  44. Holkers M, Maggio I, Liu J, Janssen JM, Miselli F. 44.  et al. 2013. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells. Nucleic Acids Res. 41:e63 [Google Scholar]
  45. Horvath P, Barrangou R. 45.  2010. CRISPR/Cas, the immune system of bacteria and archaea. Science 327:167–70 [Google Scholar]
  46. Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF. 46.  et al. 2013. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. PNAS 110:15644–49 [Google Scholar]
  47. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S. 47.  et al. 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31:827–32 [Google Scholar]
  48. Huang H, Zheng G, Jiang W, Hu H, Lu Y. 48.  2015. One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces. Acta Biochim. Biophys. Sin. 47:231–43 [Google Scholar]
  49. Huo Y, Nam KH, Ding F, Lee H, Wu L. 49.  et al. 2014. Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation. Nat. Struct. Mol. Biol. 21:771–77 [Google Scholar]
  50. Jackson RN, Golden SM, van Erp PB, Carter J, Westra ER. 50.  et al. 2014. Crystal structure of the CRISPR RNA–guided surveillance complex from Escherichia coli. Science 345:1473–79 [Google Scholar]
  51. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. 51.  2013. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31:233–39 [Google Scholar]
  52. Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. 52.  2015. Multigene editing in the Escherichia coli genome using the CRISPR-Cas9 system. Appl. Environ. Microbiol. 812506–14
  53. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 53.  2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–21 [Google Scholar]
  54. Joung JK, Sander JD. 54.  2013. TALENs: a widely applicable technology for targeted genome editing. Nat. Rev. Mol. Cell Biol. 14:49–55 [Google Scholar]
  55. Juillerat A, Dubois G, Valton J, Thomas S, Stella S. 55.  et al. 2014. Comprehensive analysis of the specificity of transcription activator-like effector nucleases. Nucleic Acids Res. 42:5390–402 [Google Scholar]
  56. Kim S, Kim D, Cho SW, Kim J, Kim J-S. 56.  2014. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24:1012–19 [Google Scholar]
  57. Kiro R, Shitrit D, Qimron U. 57.  2014. Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system. RNA Biol. 11:42–44 [Google Scholar]
  58. Koike-Yusa H, Li Y, Tan EP, del Castillo Velasco-Herrera M, Yusa K. 58.  2014. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32:267–73 [Google Scholar]
  59. Kondo S, Ueda R. 59.  2013. Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics 195:715–21 [Google Scholar]
  60. Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO. 60.  et al. 2014. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517:583–88 [Google Scholar]
  61. Kuscu C, Arslan S, Singh R, Thorpe J, Adli M. 61.  2014. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat. Biotechnol. 32:677–83 [Google Scholar]
  62. Li D, Qiu Z, Shao Y, Chen Y, Guan Y. 62.  et al. 2013. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat. Biotechnol. 31:681–83 [Google Scholar]
  63. Li W, Teng F, Li T, Zhou Q. 63.  2013. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat. Biotechnol. 31:684–86 [Google Scholar]
  64. Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN. 64.  2014. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345:1184–88 [Google Scholar]
  65. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK. 65.  2013. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10:977–79 [Google Scholar]
  66. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM. 66.  et al. 2008. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol. Cell 31:294–301 [Google Scholar]
  67. Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. 67.  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. 1:7 [Google Scholar]
  68. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E. 68.  et al. 2011. Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9:467–77 [Google Scholar]
  69. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M. 69.  et al. 2013. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31:833–38 [Google Scholar]
  70. Mali P, Yang L, Esvelt KM, Aach J, Guell M. 70.  et al. 2013. RNA-guided human genome engineering via Cas9. Science 339:823–26 [Google Scholar]
  71. Manica A, Zebec Z, Steinkellner J, Schleper C. 71.  2013. Unexpectedly broad target recognition of the CRISPR-mediated virus defence system in the archaeon Sulfolobus solfataricus. Nucleic Acids Res. 41:10509–17 [Google Scholar]
  72. Manica A, Zebec Z, Teichmann D, Schleper C. 72.  2011. In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon. Mol. Microbiol. 80:481–91 [Google Scholar]
  73. Marraffini LA, Sontheimer EJ. 73.  2008. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322:1843–45 [Google Scholar]
  74. Marraffini LA, Sontheimer EJ. 74.  2010. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463:568–71 [Google Scholar]
  75. Martel B, Moineau S. 75.  2014. CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages. Nucleic Acids Res. 42:9504–13 [Google Scholar]
  76. Mojica FJ, Díez-Villaseñor C, García-Martinez J, Almendros C. 76.  2009. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155:733–40 [Google Scholar]
  77. Mojica FJ, Díez-Villaseñor C, García-Martinez J, Soria E. 77.  2005. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60:174–82 [Google Scholar]
  78. Moscou MJ, Bogdanove AJ. 78.  2009. A simple cipher governs DNA recognition by TAL effectors. Science 326:1501 [Google Scholar]
  79. Mulepati S, Bailey S. 79.  2013. In vitro reconstitution of an Escherichia coli RNA-guided immune system reveals unidirectional, ATP-dependent degradation of DNA target. J. Biol. Chem. 288:22184–92 [Google Scholar]
  80. Mulepati S, Heroux A, Bailey S. 80.  2014. Crystal structure of a CRISPR RNA–guided surveillance complex bound to a ssDNA target. Science 345:1479–84 [Google Scholar]
  81. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI. 81.  et al. 2014. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156:935–49 [Google Scholar]
  82. Niu Y, Shen B, Cui Y, Chen Y, Wang J. 82.  et al. 2014. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156:836–43 [Google Scholar]
  83. Oh JH, van Pijkeren JP. 83.  2014. CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res. 42:e131 [Google Scholar]
  84. Paddison PJ, Silva JM, Conklin DS, Schlabach M, Li M. 84.  et al. 2004. A resource for large-scale RNA-interference-based screens in mammals. Nature 428:427–31 [Google Scholar]
  85. Mandal PK, Ferreira LM, Collins R, Meissner TB, Boutwell CL. 85.  et al. 2014. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15:643–52 [Google Scholar]
  86. Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. 86.  2013. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31:839–43 [Google Scholar]
  87. Peng W, Feng M, Feng X, Liang YX, She Q. 87.  2014. An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference. Nucleic Acids Res. 43:406–17 [Google Scholar]
  88. Pennisi E. 88.  2013. The CRISPR craze. Science 341:833–36 [Google Scholar]
  89. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM. 89.  et al. 2013. RNA-guided gene activation by CRISPR-Cas9–based transcription factors. Nat. Methods 10:973–76 [Google Scholar]
  90. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L. 90.  et al. 2014. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159:440–55 [Google Scholar]
  91. Plessis A, Perrin A, Haber JE, Dujon B. 91.  1992. Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics 130:451–60 [Google Scholar]
  92. Pourcel C, Salvignol G, Vergnaud G. 92.  2005. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151:653–63 [Google Scholar]
  93. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS. 93.  et al. 2013. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–83 [Google Scholar]
  94. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S. 94.  et al. 2013. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154:1380–89 [Google Scholar]
  95. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS. 95.  et al. 2015. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–91 [Google Scholar]
  96. Richardson C, Moynahan ME, Jasin M. 96.  1998. Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. Genes Dev. 12:3831–42 [Google Scholar]
  97. Rouet P, Smih F, Jasin M. 97.  1994. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell. Biol. 14:8096–106 [Google Scholar]
  98. Samai P, Pyenson N, Jinag W, Goldberg GW, Hatoum-Aslan A, Marraffini LA. 98.  2015. Co-transcriptional DNA and RNA cleavage during type III CRISPR-Cas immunity. Cell 161:1164–74 [Google Scholar]
  99. Sander JD, Joung JK. 99.  2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32:347–55 [Google Scholar]
  100. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. 100.  2011. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39:9275–82 [Google Scholar]
  101. Sashital DG, Wiedenheft B, Doudna JA. 101.  2012. Mechanism of foreign DNA selection in a bacterial adaptive immune system. Mol. Cell 46:606–15 [Google Scholar]
  102. Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER. 102.  et al. 2011. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. PNAS 108:10098–103 [Google Scholar]
  103. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA. 103.  et al. 2014. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343:84–87 [Google Scholar]
  104. Shen B, Zhang J, Wu H, Wang J, Ma K. 104.  et al. 2013. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 23:720–23 [Google Scholar]
  105. Shuman S, Glickman MS. 105.  2007. Bacterial DNA repair by non-homologous end joining. Nat. Rev. Microbiol. 5:852–61 [Google Scholar]
  106. Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. 106.  et al. 2011. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J. 30:1335–42 [Google Scholar]
  107. Smith J, Grizot S, Arnould S, Duclert A, Epinat JC. 107.  et al. 2006. A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Res. 34:e149 [Google Scholar]
  108. Sokolowski RD, Graham S, White MF. 108.  2014. Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system. Nucleic Acids Res. 42:6532–41 [Google Scholar]
  109. Staals RH, Zhu Y, Taylor DW, Kornfeld JE, Sharma K. 109.  et al. 2014. RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus. Mol. Cell 56:518–30 [Google Scholar]
  110. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA. 110.  2014. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507:62–67 [Google Scholar]
  111. Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y. 111.  et al. 2015. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33:102–6 [Google Scholar]
  112. Szczelkun MD, Tikhomirova MS, Sinkunas T, Gasiunas G, Karvelis T. 112.  et al. 2014. Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. PNAS 111:9798–803 [Google Scholar]
  113. Tamulaitis G, Kazlauskiene M, Manakova E, Venclovas C, Nwokeoji AO. 113.  et al. 2014. Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus. Mol. Cell 56:506–17 [Google Scholar]
  114. Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD. 114.  2014. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:635–46 [Google Scholar]
  115. Tang TH, Bachellerie JP, Rozhdestvensky T, Bortolin ML, Huber H. 115.  et al. 2002. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. PNAS 99:7536–41 [Google Scholar]
  116. Tang TH, Polacek N, Zywicki M, Huber H, Brugger K. 116.  et al. 2005. Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus. Mol. Microbiol. 55:469–81 [Google Scholar]
  117. Terns MP, Terns RM. 117.  2011. CRISPR-based adaptive immune systems. Curr. Opin. Microbiol. 14:321–27 [Google Scholar]
  118. Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V. 118.  et al. 2014. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32:569–76 [Google Scholar]
  119. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM. 119.  et al. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435:646–51 [Google Scholar]
  120. Wan H, Feng C, Teng F, Yang S, Hu B. 120.  et al. 2014. One-step generation of p53 gene biallelic mutant cynomolgus monkey via the CRISPR/Cas system. Cell Res. 25:258–61 [Google Scholar]
  121. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW. 121.  et al. 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–18 [Google Scholar]
  122. Wang T, Wei JJ, Sabatini DM, Lander ES. 122.  2014. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84 [Google Scholar]
  123. Wang Y, Zhang ZT, Seo SO, Choi K, Lu T. 123.  et al. 2015. Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system. J. Biotechnol. 200:1–5 [Google Scholar]
  124. Webber P. 124.  2014. Does CRISPR-Cas open new possibilities for patents or present a moral maze?. Nat. Biotechnol. 32:331–33 [Google Scholar]
  125. Westra ER, van Erp PB, Kunne T, Wong SP, Staals RH. 125.  et al. 2012. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol. Cell 46:595–605 [Google Scholar]
  126. Weterings E, Chen DJ. 126.  2008. The endless tale of non-homologous end-joining. Cell Res. 18:114–24 [Google Scholar]
  127. Wiedenheft B, Sternberg SH, Doudna JA. 127.  2012. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–38 [Google Scholar]
  128. Wiedenheft B, van Duijn E, Bultema J, Waghmare S, Zhou K. 128.  et al. 2011. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. PNAS 108:10092–97 [Google Scholar]
  129. Wu X, Scott DA, Kriz AJ, Chiu AC, Hsu PD. 129.  et al. 2014. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32:670–76 [Google Scholar]
  130. Wu Y, Liang D, Wang Y, Bai M, Tang W. 130.  et al. 2013. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13:659–62 [Google Scholar]
  131. Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. 131.  2013. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154:1370–79 [Google Scholar]
  132. Yin H, Xue W, Chen S, Bogorad RL, Benedetti E. 132.  et al. 2014. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32:551–53 [Google Scholar]
  133. Zebec Z, Manica A, Zhang J, White MF, Schleper C. 133.  2014. CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus. Nucleic Acids Res. 42:5280–88 [Google Scholar]
  134. Zhang J, Rouillon C, Kerou M, Reeks J, Brugger K. 134.  et al. 2012. Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol. Cell 45:303–13 [Google Scholar]
  135. Zhao H, Sheng G, Wang J, Wang M, Bunkoczi G. 135.  et al. 2014. Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli. Nature 515:147–50 [Google Scholar]
  136. Zhao Y, Dai Z, Liang Y, Yin M, Ma K. 136.  et al. 2014. Sequence-specific inhibition of microRNA via CRISPR/CRISPRi system. Sci. Rep. 4:3943 [Google Scholar]
  137. Zhou Y, Zhu S, Cai C, Yuan P, Li C. 137.  et al. 2014. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509:487–89 [Google Scholar]

Data & Media loading...

  • Article Type: Review Article
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error