Система CRISPR/Cas9 – универсальный инструмент геномной инженерии

Система CRISPR/Cas9 была изначально описана как механизм адаптивного иммунитета архей и бактерий, однако после ряда модификаций она нашла активное применение в генной инженерии, благодаря способности вносить направленный ДНК-разрыв с помощью короткого программируемого 20-нуклеотидного района в направляющей молекуле РНК (single guide RNA, sgРНК). Обзор посвящен современным приложениям системы CRISPR/Cas9 в генной инженерии. В первой его части описан основной механизм действия CRISPR/Cas9, особо уделено внимание причинам неспецифической активности Cas9 (off-targets). Она выражается в связывании комплексом Cas9-sgРНК нецелевых геномных участков, имеющих лишь частичную гомологию с sgРНК, что может приводить к нежелательным мутациям в геноме. В обзоре обсуждаются недавние улучшения специфичности связывания Cas9 и подходов по расширению функций CRISPR/Cas9 для трансгенеза. Популярность системы CRISPR/Cas9 в основном обусловлена ее выдающимся потенциалом для генной терапии и геномной инженерии, и последние достижения в этих областях представлены в нашем обзоре. В частности, CRISPR/Cas9 была недавно использована для контроля заражения клеток ВИЧ (вирусом иммунодефицита человека) и исправления генетических нарушений, таких как мышечная дистрофия Дюшенна и пигментный ретинит, на культурах клеток и животных моделях. Программируемость CRISPR/Cas9 облегчает создание трансгенных организмов с направленными генными мутациями, встройками генов и крупными хромосомными перестройками. Система CRISPR/Cas9 оказалась особенно востребованной для пронуклеарной микроинъекции при получении трансгенных сельскохозяйственных животных в биотехнологии. Один из разделов обзора посвящен генетическим скринингам на основе CRISPR/Cas9, которые приводят к высокоэффективной идентификации новых генов и генных сетей во многих биологических процессах. Наконец, в обзоре рассматривается технология генных драйверов, основанная на СRISPR/Cas9, которая представляет собой мощный инструмент для модификации экосистем в обозримом будущем.

СRISPR/Cas9, редактирование генома, трансгенез, клеточная терапия, мутагенная цепная реакция

1. Alphey L. Can CRISPR-Cas9 gene drives curb malaria? Nat. Biotechnol. 2016;34(2):149-150.

2. Anders C., Niewoehner O., Duerst A., Jinek M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014;513(7519):569-573.

3. Bakondi B., Lv W., Lu B., Jones M.K., Tsai Y., Kim K.J., Levy R., Akhtar A.A., Breunig J.J., Svendsen C.N., Wang S. In vivo CRISPR/Cas9 gene editing corrects retinal dystrophy in the S334ter-3 rat model of autosomal dominant retinitis pigmentosa. Mol. Ther.: J. Am. Soc. Gene Ther. 2015;24(3):556-563.

4. Barrangou R., Fremaux C., Deveau H., Richards M., Boyaval P., Moineau S., Romero D.A., Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes. Science (N.Y.). 2007; 315(5819):1709-1712.

5. Bassett A.R., Tibbit C., Ponting C.P., Liu J.L. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 2013;4(1):220-228.

6. Bassuk A.G., Zheng A., Li Y., Tsang S.H., Mahajan V.B. Precision medicine: genetic repair of retinitis pigmentosa in patient-derived stem cells. Sci. Rep. 2016;6:19969.

7. Bauer D.E., Canver M.C., Orkin S.H. Generation of genomic deletions in mammalian cell lines via CRISPR/Cas9. J. Vis. Exp. 2014;83:e52118.

8. Bolukbasi M.F., Gupta A., Wolfe S.A. Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery. Nat. Methods. 2016; 13(1):37-40.

9. Boutros M., Ahringer J. The art and design of genetic screens: RNA interference. Nat. Rev. Genet. 2008;9(7):554-566.

10. Brandl C., Ortiz O., Rottig B., Wefers B., Wurst W., Kuhn R. Creation of targeted genomic deletions using TALEN or CRISPR/Cas nuclease pairs in one-cell mouse embryos. FEBS Open Bio. 2015;5:26-35.

11. Burt A. Site-specific selfish genes as tools for the control and genetic engineering of natural populations. Proc. Roy. Soc. Lond. 2003;270(1518):921-928.

12. Canver M.C., Bauer D.E., Dass A., Yien Y.Y., Chung J., Masuda T., Maeda T., Paw B.H., Orkin S.H. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/cas9 nuclease system in mammalian cells. J. Biol. Chem. 2014;289(31):21312-21324.

13. Champer J., Buchman A., Akbari O.S. Cheating evolution: engineering gene drives to manipulate the fate of wild populations. Nat. Rev. Genet. 2016;17(3):146-159.

14. Chen B., Gilbert L.A., Cimini B.A., Schnitzbauer J., Zhang W., Li G.W., Park J., Blackburn E.H., Weissman J.S., Qi L.S., Huang B. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell. 2013;155(7):1479-1491.

15. Chen S., Sanjana N.E., Zheng K., Shalem O., Lee K., Shi X., Scott D.A., Song J., Pan J.Q., Weissleder R., Lee H., Zhang F., Sharp P.A. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell. 2015;160(6):1246-1260.

16. Cheng A.W., Wang H., Yang H., Shi L., Katz Y., Theunissen T.W., Rangarajan S., Shivalila C.S., Dadon D.B., Jaenisch R. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 2013;23(10):1163-1171.

17. Cho S.W., Kim S., Kim Y., Kweon J., Kim H.S., Bae S., Kim J.-S. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 2014;24(1):132-141.

18. Choi P.S., Meyerson M. Targeted genomic rearrangements using CRISPR/Cas technology. Nat. Commun. 2014;5:3728.

19. Chu V.T., Weber T., Wefers B., Wurst W., Sander S., Rajewsky K., Kühn R. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 2015;33(5):543-548.

20. Cong L., Ran F.A., Cox D., Lin S., Barretto R., Habib N., Hsu P.D., Wu X., Jiang W., Marraffini L.A., Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121): 819-823.

21. Costello M.J., May R.M., Stork N.E. Can we name Earth’s species before they go extinct? Science. 2013;339(6118):413-416.

22. Cox D.B.T., Platt R.J., Zhang F. Therapeutic genome editing: prospects and challenges. Nat. Med. 2015;21(2):121-131.

23. Crispo M., Mulet A.P., Tesson L., Barrera N., Cuadro F., dos SantosNeto P.C., Nguyen T.H., Crénéguy A., Brusselle L., Anegón I., Menchaca A. Efficient generation of myostatin knock-out sheep using CRISPR/Cas9 technology and microinjection into zygotes. PloS ONE. 2015;10(8):e0136690.

24. Cyranoski D. Embryo editing divides scientists. Nature. 2015;519 (7543):272.

25. Davis K.M., Pattanayak V., Thompson D.B., Zuris J.A., Liu D.R. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat. Chem. Biol. 2015;11(5):316-318.

26. Deredec A., Burt A., Godfray H.C.J. The population genetics of using homing endonuclease genes in vector and pest management. Genetics. 2008;179(4):2013-2026.

27. Dianov G.L., Hübscher U. Mammalian base excision repair: the forgotten archangel. Nucl. Acids Res. 2013;41(6):3483-3490.

28. Ding Q., Strong A., Patel K.M., Ng S.L., Gosis B.S., Regan S.N., Cowan C.A., Rader D.J., Musunuru K. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ. Res. 2014;115(5):488-492.

29. Doench J.G., Fusi N., Sullender M., Hegde M., Vaimberg E.W., Donovan K.F., Smith I., Tothova Z., Wilen C., Orchard R., Virgin H.W., Listgarten J., Root D.E. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 2016;34(2):184-191.

30. Elliott B., Richardson C., Winderbaum J., Nickoloff J.A., Jasin M. Gene conversion tracts from double-strand break repair in mammalian cells. Mol. Cell. Biol. 1998;18(1):93-101.

31. Essletzbichler P., Konopka T., Santoro F., Chen D., Gapp B.V., Kralovics R., Brummelkamp T.R., Nijman S.M.B., Bürckstümmer T. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res. 2014;24(12):2059-2065.

32. Fei J.F., Schuez M., Tazaki A., Taniguchi Y., Roensch K., Tanaka E.M. CRISPR-mediated genomic deletion of Sox2 in the axolotl shows a requirement in spinal cord neural stem cell amplification during tail regeneration. Stem Cell Reports. 2014;3(3):444-459.

33. Findlay G.M., Boyle E.A., Hause R.J., Klein J.C., Shendure J. Saturation editing of genomic regions by multiplex homology-directed repair. Nature. 2014;513(7516):120-123.

34. Flowers J.J., He S., Malfatti S., del Rio T.G., Tringe S.G., Hugenholtz P., McMahon K.D. Comparative genomics of two “Candidatus Accumulibacter” clades performing biological phosphorus removal. ISME J. 2013;7(12):2301-2314.

35. Frock R.L., Hu J., Meyers R.M., Ho Y.-J., Kii E., Alt F.W. Genomewide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 2015;33(2):179-186.

36. Fu Y., Foden J.A., Khayter C., Maeder M.L., Reyon D., Joung J.K., Sander J.D. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 2013;31(9): 822-826.

37. Fu Y., Sander J.D., Reyon D., Cascio V.M., Joung J.K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 2014;32(3):279-284.

38. Fujihara Y., Ikawa M. CRISPR/Cas9-based genome editing in mice by single plasmid injection. Method. Enzymol. 2014;546:319-336.

39. Fujii W., Kawasaki K., Sugiura K., Naito K. Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucl. Acids Res. 2013;41(20):e187.

40. Gantz V.M., Bier E. The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Science. 2015; 348(6233):442-444.

41. Gantz V.M., Jasinskiene N., Tatarenkova O., Fazekas A., Macias V.M., Bier E., James A.A. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proc. Natl Acad. Sci. 2015;112(49):E6736-E6743.

42. Geisinger J.M., Turan S., Hernandez S., Spector L.P., Calos M.P. In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining. Nucl. Acids Res. 2016;44(8):e76.

43. Gonzales A.P.W., Yeh J.R. Cas9-based genome editing in Zebrafish. Methods Enzymol. 2014;546:377-413.

44. González F., Zhu Z., Shi Z.-D., Lelli K., Verma N., Li Q.V, Huangfu D. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell. 2014;15(2):215-226.

45. Graham D.B., Root D.E. Resources for the design of CRISPR gene editing experiments. Genome Biol. 2015;16(1):260.

46. Gratz S.J., Wildonger J., Harrison M.M., O’Connor-Giles K.M. CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand. Fly. 2013;7(4):37-41.

47. Guilinger J.P., Thompson D.B., Liu D.R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014;32(6):577-582.

48. Gupta A., Hall V.L., Kok F.O., Shin M., McNulty J.C., Lawson N.D., Wolfe S.A. Targeted chromosomal deletions and inversions in zebrafish. Genome Res. 2013;23(6):1008-1017.

49. Hammond A., Galizi R., Kyrou K., Simoni A., Siniscalchi C., Katsanos D., Gribble M., Baker D., Marois E., Russell S., Burt A., Windbichler N., Crisanti A., Nolan T. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat. Biotechnol. 2015;34(1):1-8.

50. Han J., Zhang J., Chen L., Shen B., Zhou J., Hu B., Du Y., Tate P.H., Huang X., Zhang W. Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9. RNA Biology. 2014;11(7):829-835.

51. He Z., Proudfoot C., Mileham A.J., Mclaren D.G., Whitelaw C.B.A., Lillico S.G. Highly efficient targeted chromosome deletions using CRISPR/Cas9. Biotechnol. Bioeng. 2015;112(5):1060-1064.

52. Hendel A., Bak R.O., Clark J.T., Kennedy A.B., Ryan D.E., Roy S., Steinfeld I., Lunstad B.D., Kaiser R.J., Wilkens A.B., Bacchetta R., Tsalenko A., Dellinger D., Bruhn L., Porteus M.H. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 2015;33(9):985-989.

53. Hilton I.B., D’Ippolito A.M., Vockley C.M., Thakore P.I., Crawford G.E., Reddy T.E., Gersbach C.A. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 2015;33(5):510-517.

54. Himeda C.L., Jones T.I., Jones P.L. CRISPR/dCas9-mediated transcriptional inhibition ameliorates the epigenetic dysregulation at D4Z4 and represses DUX4-fl in FSH muscular dystrophy. Mol. Ther.: J. Am. Soc. Gene Ther. 2016;24(3):527-535.

55. Hou Z., Zhang Y., Propson N.E., Howden S.E., Chu L.-F., Sontheimer E.J., Thomson J.A. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl Acad. Sci. USA. 2013;110(39):15644-15649.

56. Hsu P.D., Scott D.A., Weinstein J.A., Ran F.A., Konermann S., Agarwala V., Li Y., Fine E.J., Wu X., Shalem O., Cradick T.J., Marraffini L.A., Bao G., Zhang F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 2013;31(9):827-832.

57. Hu X., Chang N., Wang X., Zhou F., Zhou X., Zhu X., Xiong J.-W. Heritable gene-targeting with gRNA/Cas9 in rats. Cell Res. 2013; 23(11):1322-1325.

58. Jain I.H., Zazzeron L., Goli R., Alexa K., Schatzman-Bone S., Dhillon H., Goldberger O., Peng J., Shalem O., Sanjana N.E., Zhang F., Goessling W., Zapol W.M., Mootha V.K. Hypoxia as a therapy for mitochondrial disease. Science. 2016;352(6281):54-61.

59. Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821.

60. Jinek M., Jiang F., Taylor D.W., Sternberg S.H., Kaya E., Ma E., Anders C., Hauer M., Zhou K., Lin S., Kaplan M., Iavarone A.T., Charpentier E., Nogales E., Doudna J.A. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science. 2014; 343(6176):1247997.

61. Kleinstiver B.P., Pattanayak V., Prew M.S., Tsai S.Q., Nguyen N.T., Zheng Z., Joung J.K. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016;529(7587): 490-495.

62. Konermann S., Brigham M.D., Trevino A.E., Joung J., Abudayyeh O.O., Barcena C., Hsu P.D., Habib N., Gootenberg J.S., Nishimasu H., Nureki O., Zhang F. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2014;517(7536):583-588.

63. Kraft K., Geuer S., Will A.J., Chan W., Paliou C., Borschiwer M., Harabula I., Wittler L., Franke M., Ibrahim D.M., Kragesteen B.K., Spielmann M., Mundlos S., Lupianez D.G., Andrey G. Deletions, inversions, duplications: Engineering of structural variants using CRISPR/Cas in mice. Cell Reports. 2015;10(5):833-839.

64. Kuscu C., Arslan S., Singh R., Thorpe J., Adli M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat. Biotechnol. 2014;32(7):677-683.

65. Lanphier E., Urnov F.D., Ehlen S.H., Werner M., Smolenski J. Don’t edit the human germ line. Nature. 2015;519:410-411.

66. Li D., Qiu Z., Shao Y., Chen Y., Guan Y., Liu M., Li Y., Gao N., Wang L., Lu X., Zhao Y., Liu M. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat. Biotechnol. 2013;31(8): 681-683.

67. Liang P., Xu Y., Zhang X., Ding C., Huang R., Zhang Z., Lv J., Xie X., Chen Y., Li Y., Sun Y., Bai Y., Songyang Z., Ma W., Zhou C., Huang J. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6(5):363-372.

68. Liao H.-K., Gu Y., Diaz A., Marlett J., Takahashi Y., Li M., Suzuki K., Xu R., Hishida T., Chang C.-J., Esteban C.R., Young J., Izpisua Belmonte J.C. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells. Nat. Commun. 2015; 6:6413.

69. Lin Y., Cradick T.J., Brown M.T., Deshmukh H., Ranjan P., Sarode N., Wile B.M., Vertino P.M., Stewart F.J., Bao G. CRISPR/ Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucl. Acids Res. 2014;42(11):7473-7485.

70. Liu X., Homma A., Sayadi J., Yang S., Ohashi J., Takumi T. Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system. Sci. Reports. 2016;6:19675.

71. Long C., Amoasii L., Mireault A.A., McAnally J.R., Li H., SanchezOrtiz E., Bhattacharyya S., Shelton J.M., Bassel-Duby R., Olson E.N. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2015; 351(6271):400-403.

72. Long C., McAnally J.R., Shelton J.M., Mireault A.A., Bassel-Duby R., Olson E.N. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014; 345(6201):1184-1188.

73. Makarova K.S., Wolf Y.I., Alkhnbashi O.S., Costa F., Shah S.A., Saunders S.J., Barrangou R., Brouns S.J.J., Charpentier E., Haft D.H., Horvath P., Moineau S., Mojica F.J.M., Terns R.M., Terns M.P., White M.F., Yakunin A.F., Garrett R.A., van der Oost J., Backofen R., Koonin E. V An updated evolutionary classification of CRISPRCas systems. Nat. Rev. Microbiol. 2015;13(11):722-736.

74. Mali P., Aach J., Stranges P.B., Esvelt K.M., Moosburner M., Kosuri S., Yang L., Church G.M. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 2013;31(9):833-838.

75. Malina A., Cameron C.J.F., Robert F., Blanchette M., Dostie J., Pelletier J. PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing. Nat. Commun. 2015;6:10124.

76. Mandal P.K., Ferreira L.M.R., Collins R., Meissner T.B., Boutwell C.L., Friesen M., Vrbanac V., Garrison B.S., Stortchevoi A., Bryder D., Musunuru K., Brand H., Tager A.M., Allen T.M., Talkowski M.E., Rossi D.J., Cowan C.A. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell. 2014;15(5):643-652.

77. Maruyama T., Dougan S.K., Truttmann M.C., Bilate A.M., Ingram J.R., Ploegh H.L. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 2015;33(5):538-542.

78. Mendel G. Experiments in plant hybridization. J. Roy. Hortic. Soc. 1865;IV(1865):3-47.

79. Mora C., Tittensor D.P., Adl S., Simpson A.G.B., Worm B. How many species are there on Earth and in the ocean? PLoS Biol. 2011; 9(8):e1001127.

80. Nagano T., Fraser P. No-nonsense functions for long noncoding RNAs. Cell. 2011;145(2):178-181.

81. Naldini L. Gene therapy returns to centre stage. Nature. 2015;526 (7573):351-360.

82. Nelson C.E., Gersbach C.A. Cas9 loosens its grip on off-target sites. Nat. Biotechnol. 2016;34(3):298-299.

83. Nelson C.E., Hakim C.H., Ousterout D.G., Thakore P.I., Moreb E.A., Rivera R.M.C., Madhavan S., Pan X., Ran F.A., Yan W.X., Asokan A., Zhang F., Duan D., Gersbach C.A. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2015;351(6271):403-407.

84. Ni W., Qiao J., Hu S., Zhao X., Regouski M., Yang M., Polejaeva I.A., Chen C. Efficient gene knockout in goats using CRISPR/Cas9 system. PloS ONE. 2014;9(9):e106718.

85. Nishimasu H., Cong L., Yan W.X., Ran F.A., Zetsche B., Li Y., Kurabayashi A., Ishitani R., Zhang F., Nureki O. Crystal structure of Staphylococcus aureus Cas9. Cell. 2015;162(5):1113-1126.

86. Nishimasu H., Ran F.A., Hsu P.D., Konermann S., Shehata S.I., Dohmae N., Ishitani R., Zhang F., Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014;156(5):935-949.

87. Niu Y., Shen B., Cui Y., Chen Y., Wang J., Wang L., Kang Y., Zhao X., Si W., Li W., Xiang A.P., Zhou J., Guo X., Bi Y., Si C., Hu B., Dong G., Wang H., Zhou Z., Li T., Tan T., Pu X., Wang F., Ji S., Zhou Q., Huang X., Ji W., Sha J. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 2014;156(4): 836-843.

88. O’Connell M.R., Oakes B.L., Sternberg S.H., East-Seletsky A., Kaplan M., Doudna J.A. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature. 2014;516(7530):263-266.

89. Ota S., Hisano Y., Ikawa Y., Kawahara A. Multiple genome modifications by the CRISPR/Cas9 system in zebrafish. Genes to Cells. 2014;19(7):555-564.

90. Ousterout D.G., Kabadi A.M., Thakore P.I., Majoros W.H., Reddy T.E., Gersbach C.A. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat. Commun. 2015;6:6244.

91. Parnas O., Jovanovic M., Eisenhaure T.M., Herbst R.H., Dixit A., Ye C.J., Przybylski D., Platt R.J., Tirosh I., Sanjana N.E., Shalem O., Satija R., Raychowdhury R., Mertins P., Carr S.A., Zhang F., Hacohen N., Regev A. A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell. 2015;162(3):675-686.

92. Pattanayak V., Lin S., Guilinger J.P., Ma E., Doudna J.A., Liu D.R. High-throughput profiling of off-target DNA cleavage reveals RNAprogrammed Cas9 nuclease specificity. Nat. Biotechnol. 2013; 31(9):839-843.

93. Peng J., Wang Y., Jiang J., Zhou X., Song L., Wang L., Ding C., Qin J., Liu L., Wang W., Liu J., Huang X., Wei H., Zhang P. Production of human albumin in pigs through CRISPR/Cas9-mediated knockin of human cDNA into swine albumin locus in the zygotes. Sci. Reports. 2015;5:16705.

94. Pinder J., Salsman J., Dellaire G. Nuclear domain “knock-in” screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing. Nucl. Acids Res. 2015;43(19): 9379-9392.

95. Platt R.J., Chen S., Zhou Y., Yim M.J., Swiech L., Kempton H.R., Dahlman J.E., Parnas O., Eisenhaure T.M., Jovanovic M., Graham D.B., Jhunjhunwala S., Heidenreich M., Xavier R.J., Langer R., Anderson D.G., Hacohen N., Regev A., Feng G., Sharp P.A., Zhang F. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014;159(2):440-455.

96. Ran F.A., Cong L., Yan W.X., Scott D.A., Gootenberg J.S., Kriz A.J., Zetsche B., Shalem O., Wu X., Makarova K.S., Koonin E.V., Sharp P.A., Zhang F. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520(7546):186-191.

97. Ran F.A., Hsu P.D., Lin C.-Y., Gootenberg J.S., Konermann S., Trevino A.E., Scott D.A., Inoue A., Matoba S., Zhang Y., Zhang F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154(6):1380-1389.

98. Robert F., Barbeau M., Éthier S., Dostie J., Pelletier J. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Gen. Med. 2015;7(1):93.

99. Schmid-Burgk J.L., Chauhan D., Schmidt T., Ebert T.S., Reinhardt J., Endl E., Hornung V. A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) screen identifies NEK7 as an essential component of NLRP3 inflammasome activation. J. Biol. Chem. 2016;291(1):103-109.

100. Semenova E., Jore M.M., Datsenko K.A., Semenova A., Westra E.R., Wanner B., van der Oost J., Brouns S.J., Severinov K. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl Acad. Sci. USA. 2011;108(25):10098-10103.

101. Shalem O., Sanjana N.E., Zhang F. High-throughput functional genomics using CRISPR-Cas9. Nat. Rev. Genet. 2015;16(5):299-311.

102. Shechner D.M., Hacisuleyman E., Younger S.T., Rinn J.L. Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat. Methods. 2015;12(7):664-670.

103. Slaymaker I.M., Gao L., Zetsche B., Scott D.A., Yan W.X., Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science. 2015;351(6268):84-88.

104. Song Y., Yuan L., Wang Y., Chen M., Deng J., Lv Q., Sui T., Li Z., Lai L. Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system. Cell. Mol. Life Sci. 2016;1:1-10.

105. Sternberg S.H., Redding S., Jinek M., Greene E.C., Doudna J.A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature. 2014;507(7490):62-67.

106. Tabebordbar M., Zhu K., Cheng J.K.W., Chew W.L., Widrick J.J., Yan W.X., Maesner C., Wu E.Y., Xiao R., Ran F.A., Cong L., Zhang F., Vandenberghe L.H., Church G.M., Wagers A.J., Vandenberghe H., Church G.M., Wagers A.J. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2015;351(6271):407-411.

107. Tan W., Proudfoot C., Lillico S.G., Whitelaw C.B.A. Gene targeting, genome editing: from Dolly to editors. Transgenic Res. 2016;25(3): 273-287.

108. Tanenbaum M.E., Gilbert L.A., Qi L.S., Weissman J.S., Vale R.D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014;159(3):635-646.

109. Tebas P., Stein D., Tang W.W., Frank I., Wang S.Q., Lee G., Spratt S.K., Surosky R.T., Giedlin M.A., Nichol G., Holmes M.C., Gregory P.D., Ando D.G., Kalos M., Collman R.G., Binder-Scholl G., Plesa G., Hwang W.-T., Levine B.L., June C.H. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. New Engl. J. Med. 2014;370(10):901-910.

110. Toledo C.M., Ding Y., Hoellerbauer P., Davis R.J., Basom R., Girard E.J., Lee E., Corrin P., Hart T., Bolouri H., Davison J., Zhang Q., Hardcastle J., Aronow B.J., Plaisier C.L., Baliga N.S., Moffat J., Lin Q., Li X.-N., Nam D.-H., Lee J., Pollard S.M., Zhu J., Delrow J.J., Clurman B.E., Olson J.M., Paddison P.J. Genome-wide CRISPR-Cas9 screens reveal loss of redundancy between PKMYT1 and WEE1 in glioblastoma Stem-like Cells. Cell Reports. 2015;13(11):2425-2439.

111. Tsai S.Q., Wyvekens N., Khayter C., Foden J.A., Thapar V., Reyon D., Goodwin M.J., Aryee M.J., Joung J.K. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 2014;32(6):569-576.

112. Unckless R.L., Messer P.W., Connallon T., Clark A.G. Modeling the manipulation of natural populations by the mutagenic chain reaction. Genetics. 2015;201(2):425-431.

113. Wang H., Yang H., Shivalila C.S., Dawlaty M.M., Cheng A.W., Zhang F., Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/cas-mediated genome engineering. Cell. 2013;153(4):910-918.

114. Wang X., Zhou J., Cao C., Huang J., Hai T., Wang Y., Zheng Q., Zhang H., Qin G., Miao X., Wang H., Cao S., Zhou Q., Zhao J. Efficient CRISPR/Cas9-mediated biallelic gene disruption and sitespecific knockin after rapid selection of highly active sgRNAs in pigs. Sci. Reports. 2015a;5:13348.

115. Wang Y., Zhang Z.T., Seo S.O., Choi K., Lu T., Jin Y.S., Blaschek H.P. Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system. J. Biotechnol. 2015b;200:1-5.

116. Whitworth K.M., Lee K., Benne J.A., Beaton B.P., Spate L.D., Murphy S.L., Samuel M.S., Mao J., O’Gorman C., Walters E.M., Murphy C.N., Driver J., Mileham A., McLaren D., Wells K.D., Prather R.S. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol. Reprod. 2014;91(3):78.

117. Wiedenheft B., Lander G.C., Zhou K., Jore M.M., Brouns S.J., van der Oost J., Doudna J.A., Nogales E. Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature. 2011; 477(7365):486-489.

118. Wijshake T., Baker D.J., van de Sluis B. Endonucleases: new tools to edit the mouse genome. Biochim. Bioph. Acta. 2014;1842(10): 1942-1950.

119. Wright A.V., Nunez J.K., Doudna J.A. Review biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell. 2016;164(1-2):29-44.

120. Wright A.V., Sternberg S.H., Taylor D.W., Staahl B.T., Bardales J.A., Kornfeld J.E., Doudna J.A. Rational design of a split-Cas9 enzyme complex. Proc. Natl Acad. Sci. USA. 2015;112(10):2984-2989.

121. Wu B., Luo L., Gao X.J. Cas9-triggered chain ablation of cas9 as a gene drive brake. Nat. Biotechnol. 2016;34(2):137-138.

122. Wu X., Scott D.A., Kriz A.J., Chiu A.C., Hsu P.D., Dadon D.B., Cheng A.W., Trevino A.E., Konermann S., Chen S., Jaenisch R., Zhang F., Sharp P.A. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 2014;32(7):670-676.

123. Wu Y., Liang D., Wang Y., Bai M., Tang W., Bao S., Yan Z., Li D., Li J. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell. 2013;13(6):659-662.

124. Wyvekens N., Topkar V.V., Khayter C., Joung J.K., Tsai S.Q. Dimeric CRISPR RNA-guided FokI-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing. Hum. Gene Ther. 2015; 26(7):425-431.

125. Xiao A., Wang Z., Hu Y., Wu Y., Luo Z., Yang Z., Zu Y., Li W., Huang P., Tong X., Zhu Z., Lin S., Zhang B. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucl. Acids Res. 2013;41(14):1-11.

126. Xue H.Y., Ji L.J., Gao A.M., Liu P., He J.D., Lu X.J. CRISPR-Cas9 for medical genetic screens: applications and future perspectives. J. Med. Genet. 2016;53(2):91-97.

127. Yang D., Scavuzzo M.A., Chmielowiec J., Sharp R., Bajic A., Borowiak M. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci. Reports. 2016;6:21264.

128. Yang H., Wang H., Shivalila C.S., Cheng A.W., Shi L., Jaenisch R. Onestep generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013a;154(6): 1370-1379.

129. Yang L., Grishin D., Wang G., Aach J., Zhang C.-Z., Chari R., Homsy J., Cai X., Zhao Y., Fan J.-B., Seidman C., Seidman J., Pu W., Church G. Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells. Nat. Commun. 2014;5:5507.

130. Yang L., Guell M., Byrne S., Yang J.L., De Los Angeles A., Mali P., Aach J., Kim-Kiselak C., Briggs A.W., Rios X., Huang P.Y., Daley G., Church G. Optimization of scarless human stem cell genome editing. Nucl. Acids Res. 2013b;41(19):9049-9061.

131. Yang L., Guell M., Niu D., George H., Lesha E., Grishin D., Aach J., Shrock E., Xu W., Poci J., Cortazio R., Wilkinson R.A., Fishman J.A., Church G. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science. 2015;350(6264):1101-1104.

132. Ye L., Wang J., Beyer A.I., Teque F., Cradick T.J., Qi Z., Chang J.C., Bao G., Muench M.O., Yu J., Levy J.A., Kan Y.W. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection. Proc. Natl Acad. Sci. USA. 2014;111(26):9591-9596.

133. Yen S.T., Zhang M., Deng J.M., Usman S.J., Smith C.N., Parker-Thornburg J., Swinton P.G., Martin J.F., Behringer R.R. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev. Biol. 2014;393(1):3-9.

134. Yin H., Xue W., Chen S., Bogorad R.L., Benedetti E., Grompe M., Koteliansky V., Sharp P.A., Jacks T., Anderson D.G. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 2014;32(6):551-553.

135. Young C.S., Hicks M.R., Ermolova N.V., Nakano H., Jan M., Younesi S., Karumbayaram S., Kumagai-Cresse C., Wang D., Zack J.A., Kohn D.B., Nakano A., Nelson S.F., Miceli M.C., Spencer M.J., Pyle A.D. A Single CRISPR-Cas9 deletion strategy that targets the majority of DMD patients restores dystrophin function in hiPSCderived muscle cells. Cell Stem Cell. 2016;18(4)533-540.

136. Yu C., Liu Y., Ma T., Liu K., Xu S., Zhang Y., Liu H., La Russa M., Xie M., Ding S., Qi L.S. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell. 2015;16(2): 142-147.

137. Yuan L., Sui T., Chen M., Deng J., Huang Y., Zeng J., Lv Q., Song Y., Li Z., Lai L. CRISPR/Cas9-mediated GJA8 knockout in rabbits recapitulates human congenital cataracts. Sci. Reports. 2016;6: 22024.

138. Zalatan J.G., Lee M.E., Almeida R., Gilbert L.A., Whitehead E.H., La Russa M., Tsai J.C., Weissman J.S., Dueber J.E., Qi L.S., Lim W.A. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell. 2014;160(1-2):339-350.

139. Zetsche B., Gootenberg J.S., Abudayyeh O.O., Slaymaker I.M., Makarova K.S., Essletzbichler P., Volz S.E., Joung J., van der Oost J., Regev A., Koonin E.V., Zhang F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015a;163(3): 759-771.

140. Zetsche B., Volz S.E., Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 2015b;33(2):139-142.

141. Zhang L., Jia R., Palange N.J., Satheka A.C., Togo J., An Y., Humphrey M., Ban L., Ji Y., Jin H., Feng X., Zheng Y. Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9. PLoS ONE. 2015;10(3):1-14.

142. Zheng Q., Cai X., Tan M.H., Schaffert S., Arnold C.P., Gong X., Chen C.Z., Huang S. Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. BioTechniques. 2014; 57(3):115-124.

143. Zhou J., Wang J., Shen B., Chen L., Su Y., Yang J., Zhang W., Tian X., Huang X. Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse genome targeting. FEBS J. 2014;281(7):1717-1725.