Article | 09. 2017 Vol. 1, Issue. 2
Recent progress toward precise genome editing in animals



Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University1




2017.09. 85:101


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The ability to edit a specific locus in a genome based on targeted DNA nucleases will greatly facilitate our understanding of the function of a gene in biological research, and have many practical uses in animal biotechnology and biomedicine. Recent advances, including clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9), have substantially increased the efficiency of precise genome editing in a targeted manner. Therefore, this system has been successfully adopted for highly efficient genetic modification in diverse organisms, including animals, plants and humans, and is applicable to the efficient production of transgenic animals and model animals, as well as to breeding and research involving therapeutic potential. In addition, a base editing system has been recently developed and utilized for precise base conversion. It is composed of catalytically dead Cas9 (dCas9) and cytidine deaminase that enables both highly targeted single-base changes and local sequence diversification without double-stranded DNA cleavage. In this review, we introduce the development of genome editing technologies based on targeted DNA nucleases and the recently developed base editing system, and highlight successful research achievements indicating the potential use of programmable genome editing tools in fields of animal biotechnology.



1. Bachiller D, Schellander K, Peli J, Ruther U (1991) Liposome-mediated DNA uptake by sperm cells. Mol Reprod Dev 30:194-200.  

2. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709-1712.  

3. Billon P, Bryant EE, Joseph SA, Nambiar TS, Hayward SB, Rothstein R, Ciccia A (2017) CRISPR-Mediated base editing enables efficient disruption of eukaryotic genes through induction of STOP codons. Mol Cell 67:1068-1079.  

4. Bishop JO, Smith P (1989) Mechanism of chromosomal integration of microinjected DNA. Mol Biol Med 6:283-298.  

5. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509-1512.  

6. Cabot RA, Kuhholzer B, Chan AW, Lai L, Park KW, Chong KY, Schatten G, Murphy CN, Abeydeera LR, Day BN and Prather RS (2001) Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector. Anim Biotechnol 12: 205-214.  

7. Carbery ID, Ji D, Harrington A, Brown V, Weinstein EJ, Liaw L, Cui X (2010) Targeted genome modification in mice using zinc-finger nucleases. Genetics 186:451-459.  

8. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39:e82.  

9. Chadwick AC, Wang X, Musunuru K (2017) In vivo base editing of PCSK9 (Proprotein convertase subtilisin/kexin type 9) as a therapeutic alternative to genome editing. Arterioscler Thromb Vasc Biol 37:1741-1747.  

10. Chan A, Chong K, Martinovich C, Simerly C, Schatten G (2001) Transgenic monkeys produced by retroviral gene transfer into mature oocytes. Science 291:309-312.  

11. Chen Y, Yu J, Niu Y, Qin D, Liu H, Li G, Hu Y, Wang J, Lu Y, Kang Y, Jiang Y, Wu K, Li S, Wei J, He J, Wang J, Liu X, Luo Y, Si C, Bai R, Zhang K, Liu J, Huang S, Chen Z, Wang S, Chen X, Bao X, Zhang Q, Li F, Geng R, Liang A, Shen D, Jiang T, Hu X, Ma Y, Ji W, Sun YE (2017) Modeling rett syndrome using TALEN-Edited MECP2 mutant cynomolgus monkeys. Cell 169:945-955.  

12. Cho SW, Lee J, Carroll D, Kim JS, Lee J (2013) Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195: 1177-1180.  

13. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM (1998) Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280:1256-1258.  

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

15. Conticello SG (2008) The AID/APOBEC family of nucleic acid mutators. Genome Biol 9: 229.  

16. Cooper CA, Challagulla A, Jenkins KA, Wise TG, O'Neil TE, Morris KR, Tizard ML, Doran TJ (2017) Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE). Transgenic Res 26:331-347.  

17. Cui C, Song Y, Liu J, Ge H, Li Q, Huang H, Hu L, Zhu H, Jin Y, Zhang Y (2015) Gene targeting by TALEN-induced homologous recombination in goats directs production of beta-lactoglobulin-free, high-human lactoferrin milk. Sci Rep 5:10482.  

18. Dai XM, Ryan GR, Hapel AJ, Dominguez MG, Russell RG, Kapp S, Sylvestre V, Stanley ER (2002) Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Blood 99:111-120.  

19. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602-607.  

20. Dimitrov L, Pedersen D, Ching KH, Yi H, Collarini EJ, Izquierdo S, van de Lavoir MC, Leighton PA (2016) Germline gene editing in chickens by efficient CRISPR-Mediated homologous recombination in primordial germ cells. PLoS One 11:e0154303.  

21. 21.Ding Q, Strong A, Patel KM, Ng SL, Gosis BS, Regan SN, Cowan CA, Rader DJ, Musunuru K (2014) Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ Res 115:488-492.  

22. Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4:e5553.  

23. Flisikowska T, Merkl C, Landmann M, Eser S, Rezaei N, Cui X, Kurome M, Zakhartchenko V, Kessler B, Wieland H, Rottmann O, Schmid RM, Schneider G, Kind A, Wolf E, Saur D and Schnieke A (2012) A porcine model of familial adenomatous polyposis. Gastroenterology 143:1173-1175.  

24. Gao Y, Wu H, Wang Y, Liu X, Chen L, Li Q, Cui C, Liu X, Zhang J, Zhang Y (2017) Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects. Genome Biol 18:13.  

25. Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 109: E2579-2586.  

26. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage. Nature 551:464-471.  

27. Hai T, Teng F, Guo R, Li W, Zhou Q (2014) One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res 24:372-375.  

28. Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL (1985) Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315:680-683.  

29. Han JY (2009) Germ cells and transgenesis in chickens. Comp Immunol Microbiol Infect Dis 32:61-80.  

30. Hauschild J, Petersen B, Santiago Y, Queisser AL, Carnwath JW, Lucas-Hahn A, Zhang L, Meng X, Gregory PD, Schwinzer R, Cost GJ, Niemann H (2011) Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Sci USA 108:12013-12017.  

31. Heo YT, Quan XY, Xu YN, Baek S, Choi H, Kim NH, Kim J (2015) CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells. Stem Cells Dev 24:393-402.  

32. Hess GT, Tycko J, Yao D, Bassik MC (2017) Methods and applications of CRISPR-Mediated base editing in eukaryotic genomes. Mol Cell 68:26-43.  

33. Hofmann A, Kessler B, Ewerling S, Weppert M, Vogg B, Ludwig H, Stojkovic M, Boelhauve M, Brem G, Wolf E, Pfeifer A (2003) Efficient transgenesis in farm animals by lentiviral vectors. EMBO Rep 4:1054-1060.  

34. Hongbing H, Yonghe M, Tao W, Ling L, Xiuzhi T, Rui H, Shoulong D, Kongpan L, Feng W, Ning L (2014) One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system. Front Agric Sci Eng 1:2-5.  

35. Hu R, Fan ZY, Wang BY, Deng SL, Zhang XS, Zhang JL, Han HB, Lian ZX (2017) Rapid communication: Generation of FGF5 knockout sheep via the CRISPR/Cas9 system. J Anim Sci 95:2019-2024.  

36. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227-229.  

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

38. Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet 15:321-334.  

39. Kim JS (2016) Genome editing comes of age. Nat Protoc 11:1573-1578.  

40. Kim K, Ryu SM, Kim ST, Baek G, Kim D, Lim K, Chung E, Kim S, Kim JS (2017) Highly efficient RNA-guided base editing in mouse embryos. Nat Biotechnol 35:435-437.  

41. Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA 93:1156-1160.  

42. Kling J (2009) First US approval for a transgenic animal drug. Nat Biotechnol 27:302-304.  

43. Komor A, Kim Y, Packer M, Zuris J, Liu D (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420-424.  

44. Komor A, Zhao K, Packer M, Gaudelli N, Waterbury A, Koblan L, Kim Y, Badran A, Liu D (2017) Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv 3:eaao4774.  

45. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420-424.  

46. Kubisch HM, Larson MA, Eichen PA, Wilson JM, Roberts RM (1997) Adenovirus-mediated gene transfer by perivitelline microinjection of mouse, rat, and cow embryos. Biol Reprod 56:119-124.  

47. Kunz C, Saito Y, Schar P (2009) DNA Repair in mammalian cells: Mismatched repair: variations on a theme. Cell Mol Life Sci 66:1021-1038.  

48. Kuroiwa Y, Kasinathan P, Matsushita H, Sathiyaselan J, Sullivan EJ, Kakitani M, Tomizuka K, Ishida I, Robl JM (2004) Sequential targeting of the genes encoding immunoglobulin-mu and prion protein in cattle. Nat Genet 36:775-780.  

49. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS (2002) Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295:1089-1092.  

50. Landrum MJ, Lee JM, Benson M, Brown G, Chao C, Chitipiralla S, Gu B, Hart J, Hoffman D, Hoover J, Jang W, Katz K, Ovetsky M, Riley G, Sethi A, Tully R, Villamarin-Salomon R, Rubinstein W, Maglott DR2 (2016) ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res 44:D862-868.  

51. Lavitrano M, Forni M, Varzi V, Pucci L, Bacci ML, Di Stefano C, Fioretti D, Zoraqi G, Moioli B, Rossi M, Lazzereschi D, Stoppacciaro A, Seren E, Alfani D, Cortesini R, Frati L (1997) Sperm-mediated gene transfer: production of pigs transgenic for a human regulator of complement activation. Transplant Proc 29:3508-3509.  

52. Lee HJ, Kim YM, Ono T, Han JY (2017) Genome modification technologies and their applications in avian species. Int J Mol Sci 18.  

53. Lee HJ, Lee HC, Han JY (2015) Germline modification and engineering in avian species. Mol Cells 38:743-749.  

54. Lee HJ, Lee KY, Jung KM, Park KJ, Lee KO, Suh JY, Yao Y, Nair V, Han JY (2017) Precise gene editing of chicken Na+/H+ exchange type 1 (chNHE1) confers resistance to avian leukosis virus subgroup J (ALV-J). Dev Comp Immunol 77:340-349.  

55. Lee HJ, Lee KY, Park YH, Choi HJ, Yao Y, Nair V, Han JY (2017) Acquisition of resistance to avian leukosis virus subgroup B through mutations on tvb cysteine-rich domains in DF-1 chicken fibroblasts. Vet Res 48:48.  

56. Liang P, Sun H, Sun Y, Zhang X, Xie X, Zhang J, Zhang Z, Chen Y, Ding C, Xiong Y, Ma W, Liu D, Huang J, Songyang Z (2017) Effective gene editing by high-fidelity base editor 2 in mouse zygotes. Protein Cell 8:601-611.  

57. Lillico SG, Proudfoot C, Carlson DF, Stverakova D, Neil C, Blain C, King TJ, Ritchie WA, Tan W, Mileham AJ, McLaren DG, Fahrenkrug SC, Whitelaw CB (2013) Live pigs produced from genome edited zygotes. Sci Rep 3:2847.  

58. Lim GB (2017) Gene therapy: Human genome editing in heart disease. Nat Rev Genet 18:580.  

59. Liu X, Wang Y, Guo W, Chang B, Liu J, Guo Z, Quan F, Zhang Y (2013) Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows. Nat Commun 4:2565.  

60. Liu X, Wang Y, Tian Y, Yu Y, Gao M, Hu G, Su F, Pan S, Luo Y, Guo Z, Quan F, Zhang Y (2014) Generation of mastitis resistance in cows by targeting human lysozyme gene to beta-casein locus using zinc-finger nucleases. Proc Biol Sci 281:20133368.  

61. Lorson MA, Spate LD, Samuel MS, Murphy CN, Lorson CL, Prather RS, Wells KD (2011) Disruption of the Survival Motor Neuron (SMN) gene in pigs using ssDNA. Transgenic Res 20:1293-1304.  

62. Lu Y, Zhu J (2017) Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Mol Plant 10:523-525.  

63. Luo J, Song Z, Yu S, Cui D, Wang B, Ding F, Li S, Dai Y, Li N (2014) Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases. PLoS One 9:e95225.  

64. Lutz AJ, Li P, Estrada JL, Sidner RA, Chihara RK, Downey SM, Burlak C, Wang ZY, Reyes LM, Ivary B, Yin F, Blankenship RL, Paris LL, Tector AJ (2013) Double knockout pigs deficient in N-glycolylneuraminic acid and galactose alpha-1,3-galactose reduce the humoral barrier to xenotransplantation. Xenotransplantation 20:27-35.  

65. Ma H, Marti-Gutierrez N, Park SW, Wu J, Lee Y, Suzuki K, Koski A, Ji D, Hayama T, Ahmed R, Darby H, Van Dyken C, Li Y, Kang E, Park AR, Kim D, Kim ST, Gong J, Gu Y, Xu X, Battaglia D, Krieg SA, Lee DM, Wu DH, Wolf DP, Heitner SB, Belmonte JCI, Amato P, Kim JS, Kaul S, Mitalipov S (2017) Correction of a pathogenic gene mutation in human embryos. Nature 548:413-419.  

66. Ma Y, Zhang J, Yin W, Zhang Z, Song Y, Chang X (2016) Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat Methods 13:1029-1035.  

67. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31:833-838.  

68. 68.Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823-826.  

69. Matsushita H, Sano A, Wu H, Jiao JA, Kasinathan P, Sullivan EJ, Wang Z, Kuroiwa Y (2014) Triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production. PLoS One 9:e90383.  

70. Men K, Duan X, He Z, Yang Y, Yao S, Wei Y (2017) CRISPR/Cas9-mediated correction of human genetic disease. Sci China Life Sci 60:447-457.  

71. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143-148.  

72. Moghaddassi S, Eyestone W, Bishop CE (2014) TALEN-Mediated modification of the bovine genome for large-scale production of human serum albumin. Plos One 9:e89631.  

73. Mussolino C, Cathomen T (2012) TALE nucleases: tailored genome engineering made easy. Curr Opin Biotechnol 23:644-650.  

74. Ni W, Qiao J, Hu S, Zhao X, Regouski M, Yang M, Polejaeva IA, Chen C (2014) Efficient gene knockout in goats using CRISPR/Cas9 system. PLoS One 9:e106718.  

75. Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A (2016) Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353.  

76. Niu Y, Li T, Ji W (2017) Paving the road for biomedicine: genome editing and stem cells in primates. Natl Sci Rev 4:543-549.  

77. Oishi I, Yoshii K, Miyahara D, Kagami H, Tagami T (2016) Targeted mutagenesis in chicken using CRISPR/Cas9 system. Sci Rep. 6:23980.  

78. Park KW, Cheong HT, Lai L, Im GS, Kühholzer B, Bonk A, Samuel M, Rieke A, Day BN, Murphy CN, Carter DB, Prather RS (2001) Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein. Anim Biotechnol 12:173-181.  

79. Park TS, Lee HJ, Kim KH, Kim JS, Han JY (2014) Targeted gene knockout in chickens mediated by TALENs. Proc Natl Acad Sci USA 111:12716-12721.  

80. Petters RM, Alexander CA, Wells KD, Collins EB, Sommer JR, Blanton MR, Rojas G, Hao Y, Flowers WL, Banin E, Cideciyan AV, Jacobson SG, Wong F (1997) Genetically engineered large animal model for studying cone photoreceptor survival and degeneration in retinitis pigmentosa. Nat Biotechnol 15:965-970.  

81. Porteus MH, Carroll D (2005) Gene targeting using zinc finger nucleases. Nat Biotechnol 23:967-973.  

82. Proudfoot C, Carlson DF, Huddart R, Long CR, Pryor JH, King TJ, Lillico SG, Mileham AJ, McLaren DG, Whitelaw CB, Fahrenkrug SC (2015) Genome edited sheep and cattle. Transgenic Res 24:147-153.  

83. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154:1380-1389.  

84. Reardon S (2016) Welcome to the CRISPR zoo. Nature 531:160-163.  

85. Rees HA, Komor AC, Yeh WH, Caetano-Lopes J, Warman M, Edge ASB, Liu DR (2017) Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun 8:15790.  

86. Richardson CD, Ray GJ, Bray NL, Corn JE (2016) Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes. Nat Commun 7:12463.  

87. Richt JA, Kasinathan P, Hamir AN, Castilla J, Sathiyaseelan T, Vargas F, Sathiyaseelan J, Wu H, Matsushita H, Koster J, Kato S, Ishida I, Soto C, Robl JM, Kuroiwa Y (2007) Production of cattle lacking prion protein. Nat Biotechnol 25:132-138.  

88. Robl JM, Wang Z, Kasinathan P, Kuroiwa Y (2007) Transgenic animal production and animal biotechnology. Theriogenology 67:127-133.  

89. Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, Taft PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, Kabel AC, Wohlford-Lenane CL, Davis GJ, Hanfland RA, Smith TL, Samuel M, Wax D, Murphy CN, Rieke A, Whitworth K, Uc A, Starner TD, Brogden KA, Shilyansky J, McCray PB Jr, Zabner J, Prather RS, Welsh MJ (2008) Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science 321:1837-1841.  

90. Ruan J, Xu J, Chen-Tsai RY, Li K (2017) Genome editing in livestock: Are we ready for a revolution in animal breeding industry? Transgenic Res 26:715-726.  

91. Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F (2012) A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7:171-192.  

92. Schusser B, Collarini EJ, Yi H, Izquierdo SM, Fesler J, Pedersen D, Klasing KC, Kaspers B, Harriman WD, van de Lavoir MC, Etches RJ, Leighton PA (2013) Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells. Proc Natl Acad Sci USA 110:20170-20175.  

93. Sedivy JM, Sharp PA (1989) Positive genetic selection for gene disruption in mammalian cells by homologous recombination. Proc Natl Acad Sci USA 86:227-231.  

94. Sendai Y, Sawada T, Urakawa M, Shinkai Y, Kubota K, Hoshi H, Aoyagi Y (2006) alpha1,3-Galactosyltransferase- gene knockout in cattle using a single targeting vector with loxP sequences and cre-expressing adenovirus. Transplantation 81:760-766.  

95. Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, Teramura H, Yamamoto T, Komatsu H, Miura K, Ezura H, Nishida K, Ariizumi T, Kondo A (2017) Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat Biotechnol 35:441-443.  

96. Smith J, Bibikova M, Whitby FG, Reddy AR, Chandrasegaran S, Carroll D (2000) Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res 28:3361-3369.  

97. Stern CD (2005) The chick; a great model system becomes even greater. Dev Cell 8:9-17.  

98. Sung YH, Kim JM, Kim HT, Lee J, Jeon J, Jin Y, Choi JH, Ban YH, Ha SJ, Kim CH, Lee HW, Kim JS. (2014). Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res 24:125-131.  

99. Tan W, Carlson DF, Lancto CA, Garbe JR, Webster DA, Hackett PB, Fahrenkrug SC (2013) Efficient nonmeiotic allele introgression in livestock using custom endonucleases. Proc Natl Acad Sci USA 110:16526-16531.  

100. Tanihara F, Takemoto T, Kitagawa E, Rao S, Do LT, Onishi A, Yamashita Y, Kosugi C, Suzuki H, Sembon S, Suzuki S, Nakai M, Hashimoto M, Yasue A, Matsuhisa M, Noji S, Fujimura T, Fuchimoto D, Otoi T (2016) Somatic cell reprogramming-free generation of genetically modified pigs. Sci Adv 2:e1600803.  

101. Taylor L, Carlson DF, Nandi S, Sherman A, Fahrenkrug SC, McGrew MJ (2017) Efficient TALEN-mediated gene targeting of chicken primordial germ cells. Development 144:928-934.  

102. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11:636-646.  

103. Vize PD, Michalska AE, Ashman R, Lloyd B, Stone BA, Quinn P, Wells JR, Seamark RF (1988) Introduction of a porcine growth hormone fusion gene into transgenic pigs promotes growth. J Cell Sci 90:295-300.  

104. Wall RJ, Seidel GE Jr (1992) Transgenic farm animals - A critical analysis. Theriogenology 38:337-357.  

105. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910-918.  

106. Wang K, Ouyang H, Xie Z, Yao C, Guo N, Li M, Jiao, H, Pang D (2015) Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system. Sci Rep 5: 16623.  

107. Wang S, Zhang K, Ding F, Zhao R, Li S, Li R, Xu L, Song C, Dai Y, Li N (2013) A novel promoterless gene targeting vector to efficiently disrupt PRNP gene in cattle. J Biotechnol 163:377-385.  

108. Wang X, Cai B, Zhou J, Zhu H, Niu Y, Ma B, Yu H, Lei A, Yan H, Shen Q, Shi L, Zhao X, Hua J, Huang X, Qu L, Chen Y (2016) Correction: disruption of FGF5 in cashmere goats using CRISPR/Cas9 results in more secondary hair follicles and longer fibers. PLoS One 11:e0167322.  

109. Wang X, Yu H, Lei A, Zhou J, Zeng W, Zhu H, Dong Z, Niu Y, Shi B, Cai B, Liu J, Huang S, Yan H, Zhao X, Zhou G, He X, Chen X, Yang Y, Jiang Y, Shi L, Tian X, Wang Y, Ma B, Huang X, Qu L, Chen Y (2015) Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system. Sci Rep 5:13878.  

110. Wechsler T, Meyer KE, Spratt SK, Greengard J, Santiago Y, Sproul S, Surosky R, Paschon DE, Dubois- Stringfellow N, Ando DG, Nichol G, Rebar EJ, Holmes MC (2015) ZFN-Mediated gene targeting at the albumin locus in liver results in therapeutic levels of human FIX in mice and non-human primates. Blood 126:200.  

111. Whitworth KM, Lee K, Benne JA, Beaton BP, Spate LD, Murphy SL, Samuel MS, Mao J, O'Gorman C, Walters EM, Murphy CN, Driver J, Mileham A, McLaren D, Wells KD, Prather RS (2014) Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol Reprod 91:78.  

112. Whyte JJ, Zhao J, Wells KD, Samuel MS, Whitworth KM, Walters EM, Laughlin MH, Prather RS (2011) Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs. Mol Reprod Dev 78:2.  

113. Winblad N, Lanner F (2017) Biotechnology: At the heart of gene edits in human embryos. Nature 548:398-400.  

114. Xin J, Yang H, Fan N, Zhao B, Ouyang Z, Liu Z, Zhao Y, Li X, Song J, Yang Y, Zou Q, Yan Q, Zeng Y, Lai L (2013) Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs. PLoS One 8:e84250.  

115. Xiong K, Li S, Zhang H, Cui Y, Yu D, Li Y, Sun W, Fu Y, Teng Y, Liu Z, Zhou X, Xiao P, Li J, Liu H, Chen J (2013) Targeted editing of goat genome with modular-assembly zinc finger nucleases based on activity prediction by computational molecular modeling. Mol Biol Rep 40:4251-4256.  

116. Xu L, Zhao PM, Mariano A, Han RZ (2013) Targeted myostatin gene editing in multiple mammalian species directed by a single pair of TALE nucleases. Mol Ther Nucleic Acids 2:e112.  

117. Yu B, Lu R, Yuan Y, Zhang T, Song S, Qi Z, Shao B, Zhu M, Mi F, Cheng Y (2016) Efficient TALEN-mediated myostatin gene editing in goats. BMC Dev Biol 16:26.  

118. Zhang C, Wang L, Ren G, Li Z, Ren C, Zhang T, Xu K, Zhang Z (2014) Targeted disruption of the sheep MSTN gene by engineered zinc-finger nucleases. Mol Biol Rep 41: 209-215.  

119. Zheng Q, Lin J, Huang J, Zhang H, Zhang R, Zhang X, Cao C, Hambly C, Qin G, Yao J, Song R, Jia Q, Wang X, Li Y, Zhang N, Piao Z, Ye R, Speakman JR, Wang H, Zhou Q, Wang Y, Jin W, Zhao J (2017) Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity. Proc Natl Acad Sci USA 114:E9474-E9482.  

120. Zhou W, Wan Y, Guo R, Deng M, Deng K, Wang Z, Zhang Y, Wang F (2017) Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9. PLoS One 12:e0186056.  

121. Zhou X, Xin J, Fan N, Zou Q, Huang J, Ouyang Z, Zhao Y, Zhao B, Liu Z, Lai S, Yi X, Guo L, Esteban MA, Zeng Y, Yang H, Lai L (2015) Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cell Mol Life Sci 72:1175-1184.  

122. Zhu H, Liu J, Cui C, Song Y, Ge H, Hu L, Li Q, Jin Y, Zhang Y (2016) Targeting human alpha-lactalbumin gene insertion into the goat beta-lactoglobulin locus by TALEN-Mediated homologous recombination. PLoS One 11:e0156636.  

123. Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y, Qiu JL, Wang D, Gao C (2017) Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat Biotechnol 35:438-440.