邹菊红;邹剑伟;申玉建;冯雪萍;连子童;徐建建;宋颖;胡艳;张瑜;黄艳娜;蒋钦杨;
ZOU Juhong;ZOU Jianwei;SHEN Yujian;FENG Xueping;LIAN Zitong;XU Jianjian;SONG Ying;HU yan;ZHANG Yu;HUANG Yannan;JIANG Qinyang;College of Animal Science and Technology,Guangxi University;

• 1:广西大学动物科学技术学院

摘要(Abstract)：

基因编辑技术可以从分子水平上对某个基因进行定点修饰,从而带给生物体长久的可遗传的变异,被广泛运用于生命科学研究以及临床应用。目前基因编辑的DNA核酸酶主要有锌指核酸酶(Zinc Finger Nucleases,ZFNs)、转录激活因子样效应物核酶(Transcription Activator-Like Effector Nucleases,TALENs)和成簇规则间隔的短回文重复序列/Cas9系统(Clustered Regularly Interspaced Short Palindromic Repeats,CRISPRs/Cas9)。这些DNA核酸酶通过在预定的基因组位点诱导双链断裂来提高DNA突变率而介导靶向的遗传改变,可用来改良动物性状,在家畜育种中发挥极大作用。本文综述了几种基因编辑工具的基因结构、作用原理、几种技术之间的差异及在家畜育种中的应用情况,为基因编辑技术在家畜育种中的研究提供参考。
Gene editing technology can modify a gene at the molecular level, thus bringing long-term heritable variation to organisms, which is widely used in life science research and clinical applications. At present, the main DNA nucleases edited by genes are zinc flnger nuclease(zinc flnger nucleases, ZFNs), transcriptional activator-like ribozymes(transcription activator-like effector nucleases TALENs) and clusters of regularly spaced short palindromic repeats/Cas9 system(clustered regularly interspaced short palindromic repeats, CRISPRs/Cas9). These DNA nucleases mediate targeted genetic changes by inducing double-strand breaks at predetermined genome sites to increase the rate of DNA mutation, which can be used to improve animal traits and play an important role in livestock breeding. This paper summarizes the gene structure and function principle of several gene editing tools, analyzes the differences among several techniques, and focuses on their application in livestock breeding, so as to provide reference for the research of gene editing technology in livestock breeding.

关键词(KeyWords)： 基因编辑技术;家畜;ZFNs;TALENs;CRISPR/Cas9系统
Gene editing technology;Livestock;ZFNs;TALENs;CRISPR/cas9 system

Abstract:

Keywords:

基金项目(Foundation): 广西重点研发计划项目(桂科AB18221067);; 广西牛羊产业创新团队(nycytxgxcxtd-2021-09);; 广西创新驱动发展专项(桂科AA17204052);; 崇左市科技计划项目(崇科FC2018006)

作者(Authors): 邹菊红;邹剑伟;申玉建;冯雪萍;连子童;徐建建;宋颖;胡艳;张瑜;黄艳娜;蒋钦杨;
ZOU Juhong;ZOU Jianwei;SHEN Yujian;FENG Xueping;LIAN Zitong;XU Jianjian;SONG Ying;HU yan;ZHANG Yu;HUANG Yannan;JIANG Qinyang;College of Animal Science and Technology,Guangxi University;

DOI: 10.19556/j.0258-7033.20201027-04

参考文献(References)：

• [1]任红艳,王紫君,舒畅,等.基因编辑技术在提高猪瘦肉率中的应用[J].湖北农业科学,2020,59(21):14-16.
• [2]Guha T K,Wai A,Hausner G.Programmable genome editing tools and their regulation for efficient genome engineering[J].Comput Struct Biotechnol J,2017,15:146-160.
• [3]韦永可,袁梦珂,张涌.基因编辑技术在哺乳动物上的研究应用进展[J].内蒙古大学学报（自然科学版）,2019,50(5):531-543.
• [4]李国玲,徐志谦,杨化强,等.转基因和基因编辑猪的研究进展[J].华南农业大学学报,2019,40(5):91-101.
• [5]Kim Y G,Cha J,Chandrasegaran S.Hybrid restriction enzymes:zinc flnger fusions to Fok I cleavage domain[J].Proc Natl Acad Sci USA,1996,93(3):1156-1160.
• [6]Urnov F D,Rebar E J,Holmes M C,et al.Genome editing with engineered zinc flnger nucleases[J].Nat Rev Genet,2010,11(9):636-646.
• [7]Boch J,Bonas U.Xanthomonas AvrBs3 family-type III effectors:discovery and function[J].Annu Rev Phytopathol,2010,48:419-436.
• [8]Boch J,Scholze H,Schornack S,et al.Breaking the code of DNA binding specificity of TAL-type III effectors[J].Science,2009,326(5959):1509-1512.
• [9]Cermak T,Doyle E L,Christian M,et al.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting[J].Nucleic Acids Res,2011,39(12):e82.
• [10]Christian M,Cermak T,Doyle E L,et al.Targeting DNA doublestrand breaks with TAL effector nucleases[J].Genetics,2010,186(2):757-761.
• [11]Li T,Huang S,Jiang W Z,et al.TAL nucleases (TALNs):hybrid proteins composed of TAL effectors and Fok I DNA-cleavage domain[J].Nucleic Acids Res,2011,39(1):359-372.
• [12]Li T,Huang S,Zhao X,et al.Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes[J].Nucleic Acids Res,2011,39(14):6315-6325.
• [13]Mahfouz M M,Li L,Shamimuzzaman M,et al.De novoengineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates doublestrand breaks[J].Proc Natl Acad Sci U S A,2011,108(6):2623-2628.
• [14]Miller J C,Tan S,Qiao G,et al.A TALE nuclease architecture for efficient genome editing[J].Nat Biotechnol,2011,29(2):143-148.
• [15]Joung J K,Sander J D.TALENs:a widely applicable technology for targeted genome editing[J].Nat Rev Mol Cell Biol,2013,14(1):49-55.
• [16]Moscou M J,Bogdanove A J.A simple cipher governs DNArecognition by TAL effectors[J].Science,2009,326(5959):1501.
• [17]Huang P,Xiao A,Zhou M,et al.Heritable gene targeting in zebrafish using customized TALENs[J].Nat Biotechnol,2011,29(8):699-700.
• [18]Sander J D,Cade L,Khayter C,et al.Targeted gene disruption in somatic zebrafish cells using engineered TALENs[J].Nat Biotechnol,2011,29(8):697-698.
• [19]Tesson L,Usal C,Ménoret S,et al.Knockout rats generated by embryo microinjection of TALENs[J].Nat Biotechnol,2011,29(8):695-696.
• [20]Proudfoot C,Carlson D F,Huddart R,et al.Genome edited sheep and cattle[J].Transgenic Res,2015,24(1):147-153.
• [21]Barrangou R,Fremaux C,Deveau H,et al.CRISPR provides acquired resistance against viruses in prokaryotes[J].Science,2007,315(5819):1709-1712.
• [22]Makarova K S,Wolf Y I,Alkhnbashi O S,et al.An updated evolutionary classiflcation of CRISPR-Cas systems[J].Nat Rev Microbiol,2015,13(6):722-736.
• [23]Jung T Y,An Y,Park K H,et al.Crystal structure of the Csm1subun it of the Csm complx and its singlest randed DNAspecific nuclease activity[J].Structure,2015,23(4):782-790.
• [24]李尚,石岗,冯韶华,等.CRISPR/Cas9系统在猪基因编辑中的研究进展[J].中国畜牧杂志,2019,55(12):27-32.
• [25]Cong L,Ran F A,Cox D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-823.
• [26]Brouns S J,Jore M M,Lundgren M,et al.Small CRISPR RNAs guide antiviral defense in prokaryotes[J].Science,2008,321(5891):960-964.
• [27]Karvelis T,Gasiunas G,Miksys A,et al.crRNA and tracrRNAguide Cas9-mediated DNA interference in Streptococcus thermophiles[J].RNA Biol,2013,10(5):841-851.
• [28]Gasiunas G,Barrangou R,Horvath P,et al.Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J].Proc Natl Acad Sci U S A,2012,109(39):E2579-E2586.
• [29]Sternberg S H,Redding S,Jinek M,et al.DNA interrogation by the CRISPR RNA-guided endonuclease Cas9[J].Nature,2014,507(7490):62-67.
• [30]Zhan T,Rindtorff N,Betge J,et al.CRISPR/Cas9 for cancer research and therapy[J].Semin Cancer Biol,2019,55:106-119.
• [31]Moon S B,Kim D Y,Ko J H,et al.Recent advances in the CRISPRgenome editing tool set[J].Exp Mol Med,2019,51(11):1-11.
• [32]Hockemeyer D,Wang H,Kiani S,et al.Genetic engineering of human pluripotent cells using TALE nucleases[J].Nat Biotechnol,2011,29(8):731-734.
• [33]Defrancesco L.Move over ZFNs[J].Nat Biotechnol,2011,29(8):681-684.
• [34]Boch J,Bonas U.Xanthomonas AvrBs3 family-type III effectors:discovery and function[J].Annu Rev Phytopathol,2010,48:419-436.
• [35]Kim Y,Kweon J,Kim A,et al.A library of TAL effector nucleases spanning the human genome[J].Nat Biotechnol,2013,31(3):251-258.
• [36]Adli M.The CRISPR tool kit for genome editing and beyond[J].Nat Commun,2018,9(1):1911.
• [37]Komor A C,Kim Y B,Packer M S,et al.Programmable editing of a target base in genomic DNA without double-stranded DNAcleavage[J].Nature,2016,533(7603):420-424.
• [38]Cong L,Ran F A,Cox D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-823.
• [39]Wang H,Yang H,Shivalila C S,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J].Cell,2013,153(4):910-918.
• [40]Burkard C,Lillico S G,Reid E,et al.Precision engineering for PRRSV resistance in pigs:Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function[J].PLoS Pathog,2017,13(2):e1006206.
• [41]Whitworth K M,Rowland R R,Ewen C L,et al.Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus[J].Nat Biotechnol,2016,34(1):20-22.
• [42]Gao Y,Wu H,Wang Y,et al.Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects[J].Genome Biol,2017,18(1):13.
• [43]Lillico S G,Proudfoot C,King T J,et al.Mammalian interspecies substitution of immune modulatory alleles by genome editing[J].Sci Rep,2016,6:21645.
• [44]Liu X,Wang Y,Guo W,et al.Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows[J].Nat Commun,2013,4:2565.
• [45]Lee H J,Lee K Y,Park Y H,et al.Acquisition of resistance to avian leukosis virus subgroup B through mutations on tvb cysteine-rich domains in DF-1chicken fibroblasts[J].Vet Res,2017,48 (1):48.
• [46]Bi Y,Hua Z,Liu X,et al.Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP[J].Sci Rep,2016,6:31729.
• [47]Guo R,Wan Y,Xu D,et al.Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system[J].Sci Rep,2016,6:29855.
• [48]Proudfoot C,Carlson D F,Huddart R,et al.Genome edited sheep and cattle[J].Transgenic Res,2015,24(1):147-153.
• [49]Luo J,Song Z,Yu S,et al.Efficient generation of myostatin(MSTN) biallelic mutations in cattle using zinc flnger nucleases[J].PLoS One,2014,9(4):e95225.
• [50]Qian L,Tang M,Yang J,et al.Targeted mutations in myostatin by zinc-flnger nucleases result in double-muscled phenotype in Meishan pigs[J].Sci Rep,2015,5:14435.
• [51]Tao C,Yang Y,Li X,et al.Rapid and sensitive detection of sFAT-1 transgenic pigs by visual loop-mediated isothermal amplification[J].Appl Biochem Biotechnol,2016,179(6):938-946.
• [52]Park K E,Kaucher A V,Powell A,et al.Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2gene[J].Sci Rep,2017,7:40176.
• [53]Cui C,Song Y,Liu J,et al.Gene targeting by TALEN-induced homologous recombination in goats directs production ofβ-lactoglobulin-free,high-human lactoferrin milk[J].Sci Rep,2015,5:10482.
• [54]Oishi I,Yoshii K,Miyahara D,et al.Targeted mutagenesis in chicken using CRISPR/Cas9 system[J].Sci Rep,2016,6:23980.
• [55]Hao F,Yan W,Li X,et al.Generation of cashmere goats carrying an EDAR gene mutant using CRISPR-Cas9-mediated genome editing[J].Int J Biol Sci,2018,14(4):427-436.
• [56]Lee K,Kwon D N,Ezashi T,et al.Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency[J].Proc Natl Acad Sci U S A,2014,111(20):7260-7265.
• [57]Kang J T,Ryu J,Cho B,et al.Generation of RUNX3 knockout pigs using CRISPR/Cas9-mediated gene targeting[J].Reprod Domest Anim,2016,51(6):970-978.