The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.


Article metrics loading...

Loading full text...

Full text loading...


Literature Cited

  1. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. 1.  1987. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 169:5429–33 [Google Scholar]
  2. Jansen R, Embden JD, Gaastra W, Schouls LM. 2.  2002. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43:1565–75 [Google Scholar]
  3. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 3.  2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–21 [Google Scholar]
  4. Gasiunas G, Barrangou R, Horvath P, Siksnys V. 4.  2012. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. PNAS 109:E2579–86 [Google Scholar]
  5. Cong L, Ran FA, Cox D, Lin S, Barretto R. 5.  et al. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–23 [Google Scholar]
  6. Mali P, Yang L, Esvelt KM, Aach J, Guell M. 6.  et al. 2013. RNA-guided human genome engineering via Cas9. Science 339:823–26 [Google Scholar]
  7. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. 7.  2013. RNA-programmed genome editing in human cells. eLife 2:e00471 [Google Scholar]
  8. Mali P, Esvelt KM, Church GM. 8.  2013. Cas9 as a versatile tool for engineering biology. Nat. Methods 10:957–63 [Google Scholar]
  9. Hsu PD, Lander ES, Zhang F. 9.  2014. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–78 [Google Scholar]
  10. Doudna JA, Charpentier E. 10.  2014. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096 [Google Scholar]
  11. Mojica FJ, Diez-Villaseñor C, Soria E, Juez G. 11.  2000. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol. Microbiol. 36:244–46 [Google Scholar]
  12. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P. 12.  et al. 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–12 [Google Scholar]
  13. Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R. 13.  et al. 2010. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71 [Google Scholar]
  14. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y. 14.  et al. 2011. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602–7 [Google Scholar]
  15. Rouet P, Smih F, Jasin M. 15.  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]
  16. Rouet P, Smih F, Jasin M. 16.  1994. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. PNAS 91:6064–68 [Google Scholar]
  17. Rudin N, Sugarman E, Haber JE. 17.  1989. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics 122:519–34 [Google Scholar]
  18. Plessis A, Perrin A, Haber JE, Dujon B. 18.  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]
  19. Choulika A, Perrin A, Dujon B, Nicolas JF. 19.  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]
  20. Porteus M. 20.  2016. Genome editing: a new approach to human therapeutics. Annu. Rev. Pharmacol. Toxicol. 56:163–90 [Google Scholar]
  21. Silva G, Poirot L, Galetto R, Smith J, Montoya G. 21.  et al. 2011. Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Curr. Gene Ther. 11:11–27 [Google Scholar]
  22. Grizot S, Epinat JC, Thomas S, Duclert A, Rolland S. 22.  et al. 2010. Generation of redesigned homing endonucleases comprising DNA-binding domains derived from two different scaffolds. Nucleic Acids Res. 38:2006–18 [Google Scholar]
  23. Miller J, McLachlan AD, Klug A. 23.  1985. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 4:1609–14 [Google Scholar]
  24. Kim YG, Cha J, Chandrasegaran S. 24.  1996. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. PNAS 93:1156–60 [Google Scholar]
  25. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. 25.  2010. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11:636–46 [Google Scholar]
  26. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S. 26.  et al. 2009. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509–12 [Google Scholar]
  27. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F. 27.  et al. 2010. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:757–61 [Google Scholar]
  28. Joung JK, Sander JD. 28.  2013. TALENs: a widely applicable technology for targeted genome editing. Nat. Rev. Mol. Cell Biol. 14:49–55 [Google Scholar]
  29. Marraffini LA, Sontheimer EJ. 29.  2008. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322:1843–45 [Google Scholar]
  30. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. 30.  2005. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151:2551–61 [Google Scholar]
  31. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. 31.  2013. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31:233–39 [Google Scholar]
  32. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. 32.  2013. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41:4336–43 [Google Scholar]
  33. Li JF, Norville JE, Aach J, McCormack M, Zhang D. 33.  et al. 2013. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat. Biotechnol. 31:688–91 [Google Scholar]
  34. Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S. 34.  2013. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31:691–93 [Google Scholar]
  35. Sander JD, Joung JK. 35.  2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32:347–55 [Google Scholar]
  36. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW. 36.  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]
  37. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA. 37.  et al. 2013. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154:442–51 [Google Scholar]
  38. Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE. 38.  et al. 2015. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33:510–17 [Google Scholar]
  39. Kearns NA, Pham H, Tabak B, Genga RM, Silverstein NJ. 39.  et al. 2015. Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nat. Methods 12:401–3 [Google Scholar]
  40. Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y. 40.  et al. 2014. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159:647–61 [Google Scholar]
  41. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS. 41.  et al. 2013. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–83 [Google Scholar]
  42. Lindhout BI, Fransz P, Tessadori F, Meckel T, Hooykaas PJ, van der Zaal BJ. 42.  2007. Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins. Nucleic Acids Res. 35:e107 [Google Scholar]
  43. Miyanari Y, Ziegler-Birling C, Torres-Padilla ME. 43.  2013. Live visualization of chromatin dynamics with fluorescent TALEs. Nat. Struct. Mol. Biol. 20:1321–24 [Google Scholar]
  44. Ma H, Reyes-Gutierrez P, Pederson T. 44.  2013. Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors. PNAS 110:21048–53 [Google Scholar]
  45. Thanisch K, Schneider K, Morbitzer R, Solovei I, Lahaye T. 45.  et al. 2014. Targeting and tracing of specific DNA sequences with dTALEs in living cells. Nucleic Acids Res. 42:e38 [Google Scholar]
  46. Pederson T. 46.  2014. Repeated TALEs: visualizing DNA sequence localization and chromosome dynamics in live cells. Nucleus 5:28–31 [Google Scholar]
  47. Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W. 47.  et al. 2013. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479–91 [Google Scholar]
  48. Fujita T, Fujii H. 48.  2014. Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR. PLOS ONE 9:e103084 [Google Scholar]
  49. Fujita T, Fujii H. 49.  2013. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun. 439:132–36 [Google Scholar]
  50. Price AA, Sampson TR, Ratner HK, Grakoui A, Weiss DS. 50.  2015. Cas9-mediated targeting of viral RNA in eukaryotic cells. PNAS 112:6164–69 [Google Scholar]
  51. O'Connell MR, Oakes BL, Sternberg SH, East-Seletsky A, Kaplan M, Doudna JA. 51.  2014. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516:263–66 [Google Scholar]
  52. Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Soria E. 52.  2005. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60:174–82 [Google Scholar]
  53. Rath D, Amlinger L, Rath A, Lundgren M. 53.  2015. The CRISPR-Cas immune system: biology, mechanisms and applications. Biochimie 117:119–28 [Google Scholar]
  54. Koonin EV, Krupovic M. 54.  2015. Evolution of adaptive immunity from transposable elements combined with innate immune systems. Nat. Rev. Genet. 16:184–92 [Google Scholar]
  55. Pourcel C, Salvignol G, Vergnaud G. 55.  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]
  56. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ. 56.  et al. 2008. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–64 [Google Scholar]
  57. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA. 57.  et al. 2015. An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13:722–36 [Google Scholar]
  58. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS. 58.  et al. 2015. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–71 [Google Scholar]
  59. Shmakov S, Abudayyeh OO, Makarova KS, Wolf YI, Gootenberg JS. 59.  et al. 2015. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol. Cell 60:385–97 [Google Scholar]
  60. Wiedenheft B, Sternberg SH, Doudna JA. 60.  2012. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–38 [Google Scholar]
  61. Barrangou R, Marraffini LA. 61.  2014. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol. Cell 54:234–44 [Google Scholar]
  62. Bondy-Denomy J, Davidson AR. 62.  2014. To acquire or resist: the complex biological effects of CRISPR-Cas systems. Trends Microbiol. 22:218–25 [Google Scholar]
  63. Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Almendros C. 63.  2009. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155:733–40 [Google Scholar]
  64. Deveau H, Barrangou R, Garneau JE, Labonte J, Fremaux C. 64.  et al. 2008. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190:1390–400 [Google Scholar]
  65. Shah SA, Erdmann S, Mojica FJ, Garrett RA. 65.  2013. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol. 10:891–99 [Google Scholar]
  66. Hale CR, Zhao P, Olson S, Duff MO, Graveley BR. 66.  et al. 2009. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139:945–56 [Google Scholar]
  67. Fonfara I. Rhun A, Chylinski K, Makarova KS, Lecrivain AL. 67. , Le et al. 2014. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 42:2577–90 [Google Scholar]
  68. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S. 68.  et al. 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31:827–32 [Google Scholar]
  69. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS. 69.  et al. 2015. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–91 [Google Scholar]
  70. Nishimasu H, Cong L, Yan WX, Ran FA, Zetsche B. 70.  et al. 2015. Crystal structure of Staphylococcus aureus Cas9. Cell 162:1113–26 [Google Scholar]
  71. Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM. 71.  2013. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10:1116–21 [Google Scholar]
  72. Ma H, Naseri A, Reyes-Gutierrez P, Wolfe SA, Zhang S, Pederson T. 72.  2015. Multicolor CRISPR labeling of chromosomal loci in human cells. PNAS 112:3002–7 [Google Scholar]
  73. Horvath P, Romero DA, Coute-Monvoisin AC, Richards M, Deveau H. 73.  et al. 2008. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J. Bacteriol. 190:1401–12 [Google Scholar]
  74. Zhang Y, Heidrich N, Ampattu BJ, Gunderson CW, Seifert HS. 74.  et al. 2013. Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol. Cell 50:488–503 [Google Scholar]
  75. Briner AE, Donohoue PD, Gomaa AA, Selle K, Slorach EM. 75.  et al. 2014. Guide RNA functional modules direct Cas9 activity and orthogonality. Mol. Cell 56:333–39 [Google Scholar]
  76. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M. 76.  et al. 2013. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31:833–38 [Google Scholar]
  77. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S. 77.  et al. 2013. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154:1380–89 [Google Scholar]
  78. Shen B, Zhang W, Zhang J, Zhou J, Wang J. 78.  et al. 2014. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods 11:399–402 [Google Scholar]
  79. Trevino AE, Zhang F. 79.  2014. Genome editing using Cas9 nickases. Methods Enzymol. 546:161–74 [Google Scholar]
  80. Cho SW, Kim S, Kim Y, Kweon J, Kim HS. 80.  et al. 2014. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24:132–41 [Google Scholar]
  81. Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E. 81.  et al. 2014. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343:1247997 [Google Scholar]
  82. Jiang F, Zhou K, Ma L, Gressel S, Doudna JA. 82.  2015. A Cas9-guide RNA complex preorganized for target DNA recognition. Science 348:1477–81 [Google Scholar]
  83. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI. 83.  et al. 2014. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156:935–49 [Google Scholar]
  84. Anders C, Niewoehner O, Duerst A, Jinek M. 84.  2014. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513:569–73 [Google Scholar]
  85. Jiang F, Doudna JA. 85.  2015. The structural biology of CRISPR-Cas systems. Curr. Opin. Struct. Biol. 30:100–11 [Google Scholar]
  86. Sternberg SH, LaFrance B, Kaplan M, Doudna JA. 86.  2015. Conformational control of DNA target cleavage by CRISPR-Cas9. Nature 527:110–13 [Google Scholar]
  87. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA. 87.  2014. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507:62–67 [Google Scholar]
  88. Szczelkun MD, Tikhomirova MS, Sinkunas T, Gasiunas G, Karvelis T. 88.  et al. 2014. Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. PNAS 111:9798–803 [Google Scholar]
  89. O'Geen H, Henry IM, Bhakta MS, Meckler JF, Segal DJ. 89.  2015. A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture. Nucleic Acids Res. 43:3389–404 [Google Scholar]
  90. Wu X, Scott DA, Kriz AJ, Chiu AC, Hsu PD. 90.  et al. 2014. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32:670–76 [Google Scholar]
  91. Josephs EA, Kocak DD, Fitzgibbon CJ, McMenemy J, Gersbach CA, Marszalek PE. 91.  2015. Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage. Nucleic Acids Res. 43:8924–41 [Google Scholar]
  92. Knight SC, Xie L, Deng W, Guglielmi B, Witkowsky LB. 92.  et al. 2015. Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science 350:823–26 [Google Scholar]
  93. Lieber MR. 93.  2010. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu. Rev. Biochem. 79:181–211 [Google Scholar]
  94. Xiao-Jie L, Hui-Ying X, Zun-Ping K, Jin-Lian C, Li-Juan J. 94.  2015. CRISPR-Cas9: a new and promising player in gene therapy. J. Med. Genet. 52:289–96 [Google Scholar]
  95. Kleinstiver BP, Prew MS, Tsai SQ, Nguyen NT, Topkar VV. 95.  et al. 2015. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat. Biotechnol. 33:1293–98 [Google Scholar]
  96. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT. 96.  et al. 2015. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523:481–85 [Google Scholar]
  97. Xie K, Yang Y. 97.  2013. RNA-guided genome editing in plants using a CRISPR-Cas system. Mol. Plant 6:1975–83 [Google Scholar]
  98. Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP. 98.  2013. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. 41:e188 [Google Scholar]
  99. Niu Y, Shen B, Cui Y, Chen Y, Wang J. 99.  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]
  100. Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK. 100.  et al. 2013. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194:1029–35 [Google Scholar]
  101. Liu P, Long L, Xiong K, Yu B, Chang N. 101.  et al. 2014. Heritable/conditional genome editing in C. elegans using a CRISPR-Cas9 feeding system. Cell Res. 24:886–89 [Google Scholar]
  102. Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco JA. 102.  2013. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods 10:741–43 [Google Scholar]
  103. Dow LE. 103.  2015. Modeling disease in vivo with CRISPR/Cas9. Trends Mol. Med. 21:609–21 [Google Scholar]
  104. Blasco RB, Karaca E, Ambrogio C, Cheong TC, Karayol E. 104.  et al. 2014. Simple and rapid in vivo generation of chromosomal rearrangements using CRISPR/Cas9 technology. Cell Rep. 9:1219–27 [Google Scholar]
  105. Canver MC, Bauer DE, Dass A, Yien YY, Chung J. 105.  et al. 2014. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J. Biol. Chem. 289:21312–24 [Google Scholar]
  106. He Z, Proudfoot C, Mileham AJ, McLaren DG, Whitelaw CB, Lillico SG. 106.  2015. Highly efficient targeted chromosome deletions using CRISPR/Cas9. Biotechnol. Bioeng. 112:1060–64 [Google Scholar]
  107. Choi PS, Meyerson M. 107.  2014. Targeted genomic rearrangements using CRISPR/Cas technology. Nat. Commun. 5:3728 [Google Scholar]
  108. Kim S, Kim D, Cho SW, Kim J, Kim JS. 108.  2014. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24:1012–19 [Google Scholar]
  109. Kraft K, Geuer S, Will AJ, Chan WL, Paliou C. 109.  et al. 2015. Deletions, inversions, duplications: engineering of structural variants using CRISPR/Cas in mice. Cell Rep. pii:S2211–47 [Google Scholar]
  110. Maddalo D, Manchado E, Concepcion CP, Bonetti C, Vidigal JA. 110.  et al. 2014. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516:423–27 [Google Scholar]
  111. Torres R, Martin MC, Garcia A, Cigudosa JC, Ramirez JC, Rodriguez-Perales S. 111.  2014. Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system. Nat. Commun. 5:3964 [Google Scholar]
  112. Barrangou R, May AP. 112.  2015. Unraveling the potential of CRISPR-Cas9 for gene therapy. Expert Opin. Biol. Ther. 15:311–14 [Google Scholar]
  113. Ebina H, Misawa N, Kanemura Y, Koyanagi Y. 113.  2013. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci. Rep. 3:2510 [Google Scholar]
  114. Hu W, Kaminski R, Yang F, Zhang Y, Cosentino L. 114.  et al. 2014. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. PNAS 111:11461–66 [Google Scholar]
  115. Liu X, Hao R, Chen S, Guo D, Chen Y. 115.  2015. Inhibition of hepatitis B virus by CRISPR/Cas9 system via targeting the conserved regions of viral genome. J. Gen. Virol. 96:2252–61 [Google Scholar]
  116. Dong C, Qu L, Wang H, Wei L, Dong Y, Xiong S. 116.  2015. Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication. Antivir. Res. 118:110–17 [Google Scholar]
  117. Seeger C, Sohn JA. 117.  2014. Targeting hepatitis B virus with CRISPR/Cas9. Mol. Ther. Nucleic Acids 3:e216 [Google Scholar]
  118. Zhen S, Hua L, Takahashi Y, Narita S, Liu YH, Li Y. 118.  2014. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9. Biochem. Biophys. Res. Commun. 450:1422–16 [Google Scholar]
  119. Wang J, Quake SR. 119.  2014. RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection. PNAS 111:13157–62 [Google Scholar]
  120. Ye L, Wang J, Beyer AI, Teque F, Cradick TJ. 120.  et al. 2014. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection. PNAS 111:9591–96 [Google Scholar]
  121. Li C, Guan X, Du T, Jin W, Wu B. 121.  et al. 2015. Inhibition of HIV-1 infection of primary CD4+ T cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9. J. Gen. Virol. 96:2381–93 [Google Scholar]
  122. Tebas P, Stein D, Tang WW, Frank I, Wang SQ. 122.  et al. 2014. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N. Engl. J. Med. 370:901–10 [Google Scholar]
  123. Yi G, Choi JG, Bharaj P, Abraham S, Dang Y. 123.  et al. 2014. CCR5 gene editing of resting CD4+ T cells by transient ZFN expression from HIV envelope pseudotyped nonintegrating lentivirus confers HIV-1 resistance in humanized mice. Mol. Ther. Nucleic Acids 3:e198 [Google Scholar]
  124. Badia R, Riveira-Munoz E, Clotet B, Este JA, Ballana E. 124.  2014. Gene editing using a zinc-finger nuclease mimicking the CCR5Δ32 mutation induces resistance to CCR5-using HIV-1. J. Antimicrob. Chemother. 69:1755–59 [Google Scholar]
  125. Yin H, Xue W, Chen S, Bogorad RL, Benedetti E. 125.  et al. 2014. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32:551–53 [Google Scholar]
  126. Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN. 126.  2014. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345:1184–88 [Google Scholar]
  127. Wu Y, Zhou H, Fan X, Zhang Y, Zhang M. 127.  et al. 2015. Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell Res. 25:67–79 [Google Scholar]
  128. Wu Y, Liang D, Wang Y, Bai M, Tang W. 128.  et al. 2013. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13:659–62 [Google Scholar]
  129. Schwank G, Koo BK, Sasselli V, Dekkers JF, Heo I. 129.  et al. 2013. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13:653–58 [Google Scholar]
  130. Li HL, Gee P, Ishida K, Hotta A. 130.  2015. Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system. Methods pii:S1046–2023 [Google Scholar]
  131. Park CY, Kim DH, Son JS, Sung JJ, Lee J. 131.  et al. 2015. Functional correction of large factor VIII gene chromosomal inversions in hemophilia A patient-derived iPSCs using CRISPR-Cas9. Cell Stem Cell 17:213–20 [Google Scholar]
  132. Xie F, Ye L, Chang JC, Beyer AI, Wang J. 132.  et al. 2014. Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Res. 24:1526–33 [Google Scholar]
  133. Li HL, Fujimoto N, Sasakawa N, Shirai S, Ohkame T. 133.  et al. 2015. Precise correction of the dystrophin gene in Duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Rep. 4:143–54 [Google Scholar]
  134. Song B, Fan Y, He W, Zhu D, Niu X. 134.  et al. 2015. Improved hematopoietic differentiation efficiency of gene-corrected β-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system. Stem Cells Dev. 24:1053–65 [Google Scholar]
  135. Xu P, Tong Y, Liu XZ, Wang TT, Cheng L. 135.  et al. 2015. Both TALENs and CRISPR/Cas9 directly target the HBB IVS2-654 (C > T) mutation in β-thalassemia-derived iPSCs. Sci. Rep. 5:12065 [Google Scholar]
  136. Smith C, Abalde-Atristain L, He C, Brodsky BR, Braunstein EM. 136.  et al. 2015. Efficient and allele-specific genome editing of disease loci in human iPSCs. Mol. Ther. 23:570–77 [Google Scholar]
  137. LaFountaine JS, Fathe K, Smyth HD. 137.  2015. Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9. Int. J. Pharm. 494:180–94 [Google Scholar]
  138. Wang T, Wei JJ, Sabatini DM, Lander ES. 138.  2014. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84 [Google Scholar]
  139. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA. 139.  et al. 2014. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343:84–87 [Google Scholar]
  140. Zhou Y, Zhu S, Cai C, Yuan P, Li C. 140.  et al. 2014. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509:487–91 [Google Scholar]
  141. Sanjana NE, Shalem O, Zhang F. 141.  2014. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11:783–84 [Google Scholar]
  142. Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M. 142.  et al. 2014. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 32:1262–67 [Google Scholar]
  143. Moore JD. 143.  2015. The impact of CRISPR-Cas9 on target identification and validation. Drug Discov. Today 20:450–57 [Google Scholar]
  144. Parnas O, Jovanovic M, Eisenhaure TM, Herbst RH, Dixit A. 144.  et al. 2015. A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell 162:675–86 [Google Scholar]
  145. Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera Mdel C, Yusa K. 145.  2014. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32:267–73 [Google Scholar]
  146. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. 146.  1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–11 [Google Scholar]
  147. Taylor J, Woodcock S. 147.  2015. A perspective on the future of high-throughput RNAi screening: Will CRISPR cut out the competition or can RNAi help guide the way?. J. Biomol. Screen. 20:1040–51 [Google Scholar]
  148. Dykxhoorn DM, Lieberman J. 148.  2005. The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Annu. Rev. Med. 56:401–23 [Google Scholar]
  149. Peng R, Lin G, Li J. 149.  2016. Potential pitfalls of CRISPR/Cas9-mediated genome editing. FEBS J. 2831218–31 [Google Scholar]
  150. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D. 150.  et al. 2013. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31:822–26 [Google Scholar]
  151. O'Geen H, Yu AS, Segal DJ. 151.  2015. How specific is CRISPR/Cas9 really?. Curr. Opin. Chem. Biol. 29:72–78 [Google Scholar]
  152. Wiles MV, Qin W, Cheng AW, Wang H. 152.  2015. CRISPR-Cas9-mediated genome editing and guide RNA design. Mamm. Genome 26:501–10 [Google Scholar]
  153. Guilinger JP, Thompson DB, Liu DR. 153.  2014. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32:577–82 [Google Scholar]
  154. Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V. 154.  et al. 2014. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32:569–76 [Google Scholar]
  155. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. 155.  2016. Rationally engineered Cas9 nucleases with improved specificity. Science 351:84–88 [Google Scholar]
  156. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. 156.  2014. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32:279–84 [Google Scholar]
  157. Kim D, Bae S, Park J, Kim E, Kim S. 157.  et al. 2015. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods 12:237–43 [Google Scholar]
  158. Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR. 158.  2015. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat. Chem. Biol. 11:316–18 [Google Scholar]
  159. Chu VT, Weber T, Wefers B, Wurst W, Sander S. 159.  et al. 2015. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33:543–48 [Google Scholar]
  160. Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. 160.  2015. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33:538–42 [Google Scholar]
  161. Lin S, Staahl B, Alla RK, Doudna JA. 161.  2014. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3:e04766 [Google Scholar]
  162. Shrivastav M, De Haro LP, Nickoloff JA. 162.  2008. Regulation of DNA double-strand break repair pathway choice. Cell Res. 18:134–47 [Google Scholar]
  163. Yu C, Liu Y, Ma T, Liu K, Xu S. 163.  et al. 2015. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell 16:142–47 [Google Scholar]
  164. Liang P, Xu Y, Zhang X, Ding C, Huang R. 164.  et al. 2015. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6:363–72 [Google Scholar]
  165. Baltimore D, Berg P, Botchan M, Carroll D, Charo RA. 165.  et al. 2015. A prudent path forward for genomic engineering and germline gene modification. Science 348:36–38 [Google Scholar]
  166. Lanphier E, Urnov F, Haecker SE, Werner M, Smolenski J. 166.  2015. Don't edit the human germ line. Nature 519:410–11 [Google Scholar]
  167. Vogel G. 167.  2015. Embryo engineering alarm. Science 347:1301 [Google Scholar]
  168. Kaiser J, Normile D. 168.  2015. Embryo engineering study splits scientific community. Science 348:486–87 [Google Scholar]
  169. Pollack R. 169.  2015. Eugenics lurk in the shadow of CRISPR. Science 348:871 [Google Scholar]
  170. Bosley KS, Botchan M, Bredenoord AL, Carroll D, Charo RA. 170.  et al. 2015. CRISPR germline engineering—the community speaks. Nat. Biotechnol. 33:478–86 [Google Scholar]
  171. Mathews DJ, Chan S, Donovan PJ, Douglas T, Gyngell C. 171.  et al. 2015. CRISPR: a path through the thicket. Nature 527:159–61 [Google Scholar]
  172. Esvelt KM, Smidler AL, Catteruccia F, Church GM. 172.  2014. Concerning RNA-guided gene drives for the alteration of wild populations. eLife 3:e03401 [Google Scholar]
  173. Sinkins SP, Gould F. 173.  2006. Gene drive systems for insect disease vectors. Nat. Rev. Genet. 7:427–35 [Google Scholar]
  174. Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS. 174.  2013. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8:2180–96 [Google Scholar]
  175. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM. 175.  et al. 2013. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10:973–76 [Google Scholar]
  176. Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. 176.  2013. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41:7429–37 [Google Scholar]
  177. Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M. 177.  et al. 2013. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500:472–76 [Google Scholar]
  178. Peters JM, Silvis MR, Zhao D, Hawkins JS, Gross CA, Qi LS. 178.  2015. Bacterial CRISPR: accomplishments and prospects. Curr. Opin. Microbiol. 27:121–26 [Google Scholar]
  179. Zalatan JG, Lee ME, Almeida R, Gilbert LA, Whitehead EH. 179.  et al. 2015. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160:339–50 [Google Scholar]
  180. Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO. 180.  et al. 2015. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517:583–88 [Google Scholar]
  181. Shechner DM, Hacisuleyman E, Younger ST, Rinn JL. 181.  2015. Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat. Methods 12:664–70 [Google Scholar]
  182. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK. 182.  2013. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10:977–79 [Google Scholar]
  183. Cheng AW, Wang H, Yang H, Shi L, Katz Y. 183.  et al. 2013. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23:1163–71 [Google Scholar]
  184. Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M. 184.  et al. 2015. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12:326–28 [Google Scholar]
  185. Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD. 185.  2014. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:635–46 [Google Scholar]
  186. He S, Weintraub SJ. 186.  1998. Stepwise recruitment of components of the preinitiation complex by upstream activators in vivo. Mol. Cell. Biol. 18:2876–83 [Google Scholar]
  187. Govind CK, Yoon S, Qiu H, Govind S, Hinnebusch AG. 187.  2005. Simultaneous recruitment of coactivators by Gcn4p stimulates multiple steps of transcription in vivo. Mol. Cell. Biol. 25:5626–38 [Google Scholar]
  188. Hawkins JS, Wong S, Peters JM, Almeida R, Qi LS. 188.  2015. Targeted transcriptional repression in bacteria using CRISPR interference (CRISPRi). Methods Mol. Biol. 1311:349–62 [Google Scholar]
  189. Polstein L, Perez-Pinera P, Kocak D, Vockley C, Bledsoe P. 189.  et al. 2015. Genome-wide specificity of DNA-binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators. Genome Res. 25:1158–69 [Google Scholar]
  190. Zhao Y, Dai Z, Liang Y, Yin M, Ma K. 190.  et al. 2014. Sequence-specific inhibition of microRNA via CRISPR/CRISPRi system. Sci. Rep. 4:3943 [Google Scholar]
  191. Russa MF, Qi LS. 191.  La 2015. The new state of the art: Cas9 for gene activation and repression. Mol. Cell. Biol. 35:3800–9 [Google Scholar]
  192. Dominguez AA, Lim WA, Qi LS. 192.  2016. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 17:5–15 [Google Scholar]
  193. Lin S, Ewen-Campen B, Ni X, Housden BE, Perrimon N. 193.  2015. In vivo transcriptional activation using CRISPR/Cas9 in Drosophila. Genetics 201:433–42 [Google Scholar]
  194. Piatek A, Ali Z, Baazim H, Li L, Abulfaraj A. 194.  et al. 2015. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol. J. 13:578–89 [Google Scholar]
  195. Lowder LG, Zhang D, Baltes NJ, Paul JW 3rd, Tang X. 195.  et al. 2015. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 169:971–85 [Google Scholar]
  196. Zhang Y, Yin C, Zhang T, Li F, Yang W. 196.  et al. 2015. CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs. Sci. Rep. 5:16277 [Google Scholar]
  197. Mendenhall EM, Williamson KE, Reyon D, Zou JY, Ram O. 197.  et al. 2013. Locus-specific editing of histone modifications at endogenous enhancers. Nat. Biotechnol. 31:1133–36 [Google Scholar]
  198. Snowden AW, Gregory PD, Case CC, Pabo CO. 198.  2002. Gene-specific targeting of H3K9 methylation is sufficient for initiating repression in vivo. Curr. Biol. 12:2159–66 [Google Scholar]
  199. Maeder ML, Angstman JF, Richardson ME, Linder SJ, Cascio VM. 199.  et al. 2013. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat. Biotechnol. 31:1137–42 [Google Scholar]
  200. Rivenbark AG, Stolzenburg S, Beltran AS, Yuan X, Rots MG. 200.  et al. 2012. Epigenetic reprogramming of cancer cells via targeted DNA methylation. Epigenetics 7:350–60 [Google Scholar]
  201. Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M. 201.  et al. 2013. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500:472–76 [Google Scholar]
  202. Keung AJ, Bashor CJ, Kiriakov S, Collins JJ, Khalil AS. 202.  2014. Using targeted chromatin regulators to engineer combinatorial and spatial transcriptional regulation. Cell 158:110–20 [Google Scholar]
  203. Thakore PI, D'Ippolito AM, Song L, Safi A, Shivakumar NK. 203.  et al. 2015. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12:1143–49 [Google Scholar]
  204. Lanctôt C, Cheutin T, Cremer M, Cavalli G, Cremer T. 204.  2007. Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nat. Rev. Genet. 8:104–15 [Google Scholar]
  205. Schneider R, Grosschedl R. 205.  2007. Dynamics and interplay of nuclear architecture, genome organization, and gene expression. Genes Dev. 21:3027–43 [Google Scholar]
  206. Dixon JR, Jung I, Selvaraj S, Shen Y, Antosiewicz-Bourget JE. 206.  et al. 2015. Chromatin architecture reorganization during stem cell differentiation. Nature 518:331–36 [Google Scholar]
  207. Peric-Hupkes D, Meuleman W, Pagie L, Bruggeman SW, Solovei I. 207.  et al. 2010. Molecular maps of the reorganization of genome–nuclear lamina interactions during differentiation. Mol. Cell 38:603–13 [Google Scholar]
  208. Gall JG, Pardue ML. 208.  1969. Formation and detection of RNA–DNA hybrid molecules in cytological preparations. PNAS 63:378–83 [Google Scholar]
  209. John HA, Birnstiel ML, Jones KW. 209.  1969. RNA–DNA hybrids at the cytological level. Nature 223:582–87 [Google Scholar]
  210. Pardue ML, Gall JG. 210.  1969. Molecular hybridization of radioactive DNA to the DNA of cytological preparations. PNAS 64:600–4 [Google Scholar]
  211. Pinkel D, Straume T, Gray JW. 211.  1986. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. PNAS 83:2934–38 [Google Scholar]
  212. Pinkel D, Gray JW, Trask B, van den Engh G, Fuscoe J, van Dekken H. 212.  1986. Cytogenetic analysis by in situ hybridization with fluorescently labeled nucleic acid probes. Cold Spring Harb. Symp. Quant. Biol. 51:Pt 1151–57 [Google Scholar]
  213. Speicher MR, Gwyn Ballard S, Ward DC. 213.  1996. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat. Genet. 12:368–75 [Google Scholar]
  214. Vorsanova SG, Yurov YB, Iourov IY. 214.  2010. Human interphase chromosomes: a review of available molecular cytogenetic technologies. Mol. Cytogenet. 3:1 [Google Scholar]
  215. Riegel M. 215.  2014. Human molecular cytogenetics: from cells to nucleotides. Genet. Mol. Biol. 37:194–209 [Google Scholar]
  216. Cmarko D, Ligasova A, Koberna K. 216.  2014. Tracking DNA and RNA sequences at high resolution. Methods Mol. Biol. 1117:343–66 [Google Scholar]
  217. Hutchison NJ, Langer-Safer PR, Ward DC, Hamkalo BA. 217.  1982. In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold. J. Cell Biol. 95:609–18 [Google Scholar]
  218. Molenaar C, Wiesmeijer K, Verwoerd NP, Khazen S, Eils R. 218.  et al. 2003. Visualizing telomere dynamics in living mammalian cells using PNA probes. EMBO J. 22:6631–41 [Google Scholar]
  219. Robinett CC, Straight A, Li G, Willhelm C, Sudlow G. 219.  et al. 1996. In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J. Cell Biol. 135:1685–700 [Google Scholar]
  220. Marshall WF, Straight A, Marko JF, Swedlow J, Dernburg A. 220.  et al. 1997. Interphase chromosomes undergo constrained diffusional motion in living cells. Curr. Biol. 7:930–39 [Google Scholar]
  221. Li P, Jin H, Hoang ML, Yu HG. 221.  2011. Tracking chromosome dynamics in live yeast cells: coordinated movement of rDNA homologs and anaphase disassembly of the nucleolus during meiosis. Chromosome Res. 19:1013–26 [Google Scholar]
  222. Wang X, Kam Z, Carlton PM, Xu L, Sedat JW, Blackburn EH. 222.  2008. Rapid telomere motions in live human cells analyzed by highly time-resolved microscopy. Epigenetics Chromatin 1:4 [Google Scholar]
  223. Shelby RD, Hahn KM, Sullivan KF. 223.  1996. Dynamic elastic behavior of α-satellite DNA domains visualized in situ in living human cells. J. Cell Biol. 135:545–57 [Google Scholar]
  224. Bronstein I, Israel Y, Kepten E, Mai S, Shav-Tal Y. 224.  et al. 2009. Transient anomalous diffusion of telomeres in the nucleus of mammalian cells. Phys. Rev. Lett. 103:018102 [Google Scholar]
  225. Anton T, Bultmann S, Leonhardt H, Markaki Y. 225.  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]
  226. Deng W, Shi X, Tjian R, Lionnet T, Singer RH. 226.  2015. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells. PNAS 112:11870–75 [Google Scholar]
  227. Kuscu C, Arslan S, Singh R, Thorpe J, Adli M. 227.  2014. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat. Biotechnol. 32:677–83 [Google Scholar]
  228. Duan J, Lu G, Xie Z, Lou M, Luo J. 228.  et al. 2014. Genome-wide identification of CRISPR/Cas9 off-targets in human genome. Cell Res. 24:1009–12 [Google Scholar]
  229. Dow LE, Fisher J, O'Rourke KP, Muley A, Kastenhuber ER. 229.  et al. 2015. Inducible in vivo genome editing with CRISPR-Cas9. Nat. Biotechnol. 33:390–94 [Google Scholar]
  230. Gonzalez F, Zhu Z, Shi ZD, Lelli K, Verma N. 230.  et al. 2014. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15:215–26 [Google Scholar]
  231. Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M. 231.  2015. CRISPR-Cas9-based photoactivatable transcription system. Chem. Biol. 22:169–74 [Google Scholar]
  232. Polstein LR, Gersbach CA. 232.  2015. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11:198–200 [Google Scholar]
  233. Zetsche B, Volz SE, Zhang F. 233.  2015. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33:139–42 [Google Scholar]
  234. Wright AV, Sternberg SH, Taylor DW, Staahl BT, Bardales JA. 234.  et al. 2015. Rational design of a split-Cas9 enzyme complex. PNAS 112:2984–89 [Google Scholar]
  235. Nihongaki Y, Kawano F, Nakajima T, Sato M. 235.  2015. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat. Biotechnol. 33:755–60 [Google Scholar]
  236. Truong DJ, Kuhner K, Kuhn R, Werfel S, Engelhardt S. 236.  et al. 2015. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res. 43:6450–58 [Google Scholar]
  237. Sampson TR, Weiss DS. 237.  2013. Cas9-dependent endogenous gene regulation is required for bacterial virulence. Biochem. Soc. Trans. 41:1407–11 [Google Scholar]
  238. Sampson TR, Saroj SD, Llewellyn AC, Tzeng YL, Weiss DS. 238.  2013. A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature 497:254–57 [Google Scholar]
  239. Nelles DA, Fang MY, Aigner S, Yeo GW. 239.  2015. Applications of Cas9 as an RNA-programmed RNA-binding protein. BioEssays 37:732–39 [Google Scholar]
  240. Ma H, Wu Y, Dang Y, Choi JG, Zhang J, Wu H. 240.  2014. Pol III promoters to express small RNAs: delineation of transcription initiation. Mol. Ther. Nucleic Acids 3:e161 [Google Scholar]
  241. Hwang WY, Fu Y, Reyon D, Maeder ML, Kaini P. 241.  et al. 2013. Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLOS ONE 8:e68708 [Google Scholar]
  242. Nielsen S, Yuzenkova Y, Zenkin N. 242.  2013. Mechanism of eukaryotic RNA polymerase III transcription termination. Science 340:1577–80 [Google Scholar]
  243. Pengue G, Lania L. 243.  1996. Krüppel-associated box-mediated repression of RNA polymerase II promoters is influenced by the arrangement of basal promoter elements. PNAS 93:1015–20 [Google Scholar]
  244. Groner AC, Meylan S, Ciuffi A, Zangger N, Ambrosini G. 244.  et al. 2010. KRAB–zinc finger proteins and KAP1 can mediate long-range transcriptional repression through heterochromatin spreading. PLOS Genet. 6:e1000869 [Google Scholar]
  245. Mittler G, Stühler T, Santolin L, Uhlmann T, Kremmer E. 245.  et al. 2003. A novel docking site on Mediator is critical for activation by VP16 in mammalian cells. EMBO J. 22:6494–504 [Google Scholar]
  246. Gaj T, Epstein BE, Schaffer DV. 246.  2016. Genome engineering using adeno-associated virus: basic and clinical research applications. Mol. Ther. 24458–64 [Google Scholar]
  247. Friedland AE, Baral R, Singhal P, Loveluck K, Shen S. 247.  et al. 2015. Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol. 16:257 [Google Scholar]
  248. Crispo M, Mulet AP, Tesson L, Barrera N, Cuadro F. 248.  et al. 2015. Efficient generation of myostatin knock-out sheep using CRISPR/Cas9 technology and microinjection into zygotes. PLOS ONE 10:e0136690 [Google Scholar]
  249. Bassett AR, Tibbit C, Ponting CP, Liu JL. 249.  2013. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 4:220–28 [Google Scholar]
  250. Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T. 250.  et al. 2014. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514:380–84 [Google Scholar]
  251. Cho SW, Lee J, Carroll D, Kim JS. 251.  2013. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195:1177–80 [Google Scholar]
  252. Sung YH, Kim JM, Kim HT, Lee J, Jeon J. 252.  et al. 2014. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res. 24:125–31 [Google Scholar]
  253. Liu J, Gaj T, Yang Y, Wang N, Shui S. 253.  et al. 2015. Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells. Nat. Protoc. 10:1842–59 [Google Scholar]
  254. Schumann K, Lin S, Boyer E, Simeonov DR, Subramaniam M. 254.  et al. 2015. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. PNAS 112:10437–42 [Google Scholar]
  255. Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE. 255.  et al. 2015. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33:985–89 [Google Scholar]
  256. Liang X, Potter J, Kumar S, Zou Y, Quintanilla R. 256.  et al. 2015. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J. Biotechnol. 208:44–53 [Google Scholar]
  257. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL. 257.  et al. 2015. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33:73–80 [Google Scholar]
  258. Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H. 258.  2014. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24:1020–27 [Google Scholar]
  259. D'Astolfo DS, Pagliero RJ, Pras A, Karthaus WR, Clevers H. 259.  et al. 2015. Efficient intracellular delivery of native proteins. Cell 161:674–90 [Google Scholar]
  260. Sun W, Ji W, Hall JM, Hu Q, Wang C. 260.  et al. 2015. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew. Chem. Int. Ed. Engl. 54:12029–33 [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