基因编辑技术原理及其在动植物研究中的应用
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摘要
自人类基因组计划开展以来,越来越多生物的基因组序列得到了测定,基因的功能逐步得到鉴定.人们期望通过对基因表达的改变,来治疗人类疾病或提高生物的产量和品质.早期突变技术对基因的改变是不定向的,近年来,锌指核酸酶(ZFN)、转录激活子样效应因子核酸酶(TALEN)和CRISPR/Cas9等技术可对某个已知基因进行编辑.特别是CRISPR/Cas9技术,由于具有操作方便、效率高等优点,因此成为对基因进行定向操作的强有力工具.本文对几种基因编辑技术的原理和应用进行简要介绍和展望.
Since the launch of the Human Genome Project,more and more organisms′ genome sequences have been determined,and the function of genes has been gradually identified. Enormous endeavors have been made to treat human diseases or improve the production and quality of organisms by manipulating gene expression. Gene mutation is not site-specific by traditional mutagenesis,while technologies such as Zinic-finger nucleases(ZFN),transcription activator-like effector nucleases(TALEN)and CRISPR/Cas9 can be targeted to specific sequences. CRISPR/Cas9 system has become a powerful tool for gene editing because of its construction convenience and high efficiency. In this paper,the principle and application of several gene editing technologies were briefly introduced and prospected to help people a better understanding of gene edi-ting technology.
Since the launch of the Human Genome Project,more and more organisms′ genome sequences have been determined,and the function of genes has been gradually identified. Enormous endeavors have been made to treat human diseases or improve the production and quality of organisms by manipulating gene expression. Gene mutation is not site-specific by traditional mutagenesis,while technologies such as Zinic-finger nucleases(ZFN),transcription activator-like effector nucleases(TALEN)and CRISPR/Cas9 can be targeted to specific sequences. CRISPR/Cas9 system has become a powerful tool for gene editing because of its construction convenience and high efficiency. In this paper,the principle and application of several gene editing technologies were briefly introduced and prospected to help people a better understanding of gene edi-ting technology.
引文
[1]任志强,杨慧珍,卜华虎,等.诱变在作物遗传育种中的应用进展[J].中国农学通报,2016,32(33):125-129.REN Z Q,YANG H Z,BU H H,et al.Research progress of mutation in crop genetics breeding[J].Chinese Agricultural Science Bulletin,2016,32(33):125-129(in Chinese).
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[5]KRYSAN P J,YOUNG J C,SUSSMAN M R.T-DNA as an insertional mutagen in Arabidopsis[J].The Plant Cell,1999,11(12):2283-2290.
[6]栾维江,孙宗修.Ac/Ds标签系统与水稻功能基因组学[J].植物生理与分子生物学学报,2005,31(5):441-450.LUAN W J,SUN Z X.Ac/Ds tagging system and functional genomics in rice[J].Journal of Plant Physiology and Molecular Biology,2005,31(5):441-450(in Chinese).
[7]HIROCHIKA H,SUGIMOTO K,OTSUKI Y,et al.Retrotransposons of rice involved in mutations induced by tissue culture[J].Proc Natl Acad Sci USA,1996,93(15):7783-7788.
[8]HIROCHIKA H.Contribution of the Tos17 retrotransposon to rice functional genomics[J].Current Opinion in Plant Biology,2001,4:118-122.
[9]PORTEUS M H,CARROLL D.Gene targeting using zinc finger nucleases[J].Nature Biotechnology,2005,23(8):967-973.
[10]PAVLETICH N P,PABO C O.Zinc Finger-DNA fecognition:Crystal structure of a Zif268-DNA complex at 2.1A[J].Science,1991,252(5007):809-817.
[11]CARROLL D.Progress and prospects:Zinc-finger nucleases as gene therapy agents[J].Gene Therapy,2008,15(22):1463-1468.
[12]BOLLER T,HE S Y.Innate immunity in plants:An arms race between pattern recognition receptors in plants and effectors in microbial pathogens[J].Science,2009,324(5928):742-744.
[13]KAY S,BONAS U.How Xanthomonas typeⅢeffectors manipulate the host plant[J].Current Opinion in Microbiology,2009,12(1):37-43.
[14]BOCH J,SCHOLZE H,SCHORNACK S,et al.Breaking the code of DNA binding specificity of TAL-typeⅢeffectors[J].Science,2009,326(5959):1509-1512.
[15]SANJANA N E,CONG L,ZHOU Y,et al.A transcription activatorlike effector toolbox for genome engineering[J].Nature Protocols,2012,7(1):171-192.
[16]ZHANG F,CONG L,LODATO S,et al.Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription[J].Nature Biotechnology,2011,29(2):149-154.
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[19]HOCKEMEYER D,WANG H,KIANI S,et al.Genetic engineering of human pluripotent cells using TALE nucleases[J].Nature Biotechnology,2011,29(8):731-734.
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[21]ZHANG Y,ZHANG F,LI X,et al.Transcription activator-like effector nucleases enable efficient plant genome engineering[J].Plant Physiology,2013,161(1):20-27.
[22]SHAN Q W,WANG Y P,CHEN K L,et al.Rapid and efficient gene modification in rice and Brachypodium using TALENs[J].Molecular Plant,2013,6(4):1365-1368.
[23]ISHINO Y,SHINAGAWA H,MAKINO K,et al.Nucleotide sequence of the iap gene,responsible for alkaline phosphatase isozyme conversion in Escherichia coli,and identification of the gene product[J].Journal of Bacteriology,1987,169(12):5429-5433.
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[25]MOJICA F J M,FERRER C,JUEZ G,et al.Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloforax mediterranei and Haloferax volcanii and could be involved in replicon partitioning[J].Molecular Microbiology,1995,17(1):85-93.
[26]MOJICA F J M,D魱EZ-VILLASE N′′OR C,SORIA E,et al.Biological significance of a family of regularly spaced repeats in the genomes of Archaea,Bacteria and mitochondria[J].Molecular Microbiology,2000,36(1):244-246.
[27]JANSEN R,EMBDEN J D A,GAASTRA W,et al.Identification of genes that are associated with DNA repeats in prokaryotes[J].Molecular Microbiology,2002,43(6):1565-1575.
[28]MOJICA F J M,D魱EZ-VILLASEN′′OR C,GARC魱A-MART魱NEZ J,et al.Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements[J].Journal of Molecular Evolution,2005,60(2):174-182.
[29]POURCEL C,SALVIGNOL G,VERGNAUD G.CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA,and provide additional tools for evolutionary studies[J].Microbiology,2005,151:653-663.
[30]BOLOTIN A,QUINQUIS B,SOROKIN A,et al.Clustered regularly interspaced short palindrome repeats(CRISPRs)have spacers of extrachromosomal origin[J].Microbiology,2005,151:2551-2561.
[31]MAKAROVA K S,ARAVIND L,GRISHIN N V,et al.A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis[J].Nucleic Acids Research,2002,30(2):482-496.
[32]MAKAROVA K S,GRISHIN N V,SHABALINA S A,et al.A putative RNA-interference-based immune system in prokaryotes:Computational analysis of the predicted enzymatic machinery,functional analogies with eukaryotic RNAi,and hypothetical mechanisms of action[J].Biology Direct,2006,1(7):1-26.
[33]BARRANGOU R,FREMAUX C,DEVEAU H,et al.CRISPR provides acquired resistance against viruses in prokaryotes[J].Science,2007,315(5819):1709-1712.
[34]MOJICA F J M,D魱EZ-VILLASEN′′OR C,GARCIA-MART魱NEZ J,et al.Short motif sequences determine the targets of the prokaryotic CRISPR defence system[J].Microbiology,2009,155:733-740.
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[36]CARTE J,WANG R Y,LI H,et al.Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes[J].Gens Development,2008,22(24):3489-3496.
[37]HAURWITZ R E,JINEK M,WIEDENHEFT B,et al.Sequence-and structure-specific RNA processing by a CRISPR endonuclease[J].Science,2010,329(5997):1355-1358.
[38]DELTCHEVA E,CHYLINSKI K,SHARMA C M,et al.CRISPR RNAmaturation by trans-encoded small RNA and host factor RNaseⅢ[J].Nature,2011,471(7340):602-607.
[39]GOTTESMAN S.Dicing defence in bacteria[J].Nature,2011,471(7340):588-589.
[40]MAKAROVA K S,Haft D H,BARRANGOU R,et al.Evolution and classification of the CRISPR-Cas systems[J].Nature Reviews Microbiology,2011,9(6):467-477.
[41]JINEK M,CHYLINSKI K,FONFARA I,et al,A programmable dualRNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,2012,337(6096):816-821.
[42]GASIUNAS G,BARRANGOU R,HORVATH P,et al.Cas9-crRNAribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J].Proc Natl Acad Sci USA,2012,109(39):2579-2586.
[43]CONG L,RAN F A,COX D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-823.
[44]MALI P,YANG L H,ESVELT K M,et al.RNA-guided human genome engineering via Cas9[J].Science,2013,339(6121):823-826.
[45]HWANG W Y,FU Y,REYON D,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system[J].Nature Biotechnology,2013,31(3):227-229.
[46]BASSETT A R,TIBBIT C,PONTING CP,et al.Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system[J].Cell Reports,2013,4(1):220-228.
[47]NAKAYAMA T,FISH M B,FISHER M,et al.Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis[J].Genesis,2013,51(12):835-843.
[48]NIU Y Y,SHEN B,CUI Y Q,et al.Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos[J].Cell,2014,156(4):836-843.
[49]SHAN Q W,WANG Y P,LI J,et al.Targeted genome modification of crop plants using a CRISPR-Cas system[J].Nature Biotechnology,2013,31(8):686-688.
[50]LI J F,NORVILLE J E,AACH J,et al.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9[J].Nature Biotechnology,2013,31(8):688-691.
[51]NEKRASOV V,STASKAWICZ B,WEIGEL D,et al.Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease[J].Nature Biotechnology,2013,31(8):691-693.
[52]MAO Y F,ZHANG H,XU N F,et al.Application of the CRISPR-Cas system for efficient genome engineering in plants[J].Molecular Plant,2013,6(6):2008-2011.
[53]FENG Z Y,ZHANG B T,DING W N,et al.Efficient genome editing in plants using a CRISPR/Cas system[J].Cell Research,2013,23(10):1229-1232.
[54]MIAO J,GUO D S,ZHANG J Z,et al.Targeted mutagenesis in rice using CRISPR-Cas system[J].Cell Research,2013,23(10):1233-1236.
[55]MA X L,ZHANG Q Y,ZHU Q L,et al.A robust CRISPR/Cas9 system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants[J].Molecular Plant,2015,8(8):1274-1284.
[56]ZETSCHE B,GOOTENBERG J S,ABUDAYYEH O O,et al.Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system[J].Cell,2015,163(3):759-771.
[57]HU J H,MILLER S M,GEURTS M H,et al.Evolved Cas9 variants with broad PAM compatibility and high DNA specificity[J].Nature,2018,556(7699):57-63.
[58]杨伟,李施施,张瑄,等.基于诱导多能干细胞的基因编辑和细胞治疗[J].中国细胞生物学学报,2015,37(1):90-99.YANG W,LI S S,ZHANG X,et al.Gene editing and cell therapy based on induced pluripotent stem cells[J].Chinese Journal of Cell Biology,2015,37(1):90-99(in Chinese).
[59]季海艳,朱焕章.基因编辑技术在基因治疗中的应用进展[J].生命科学,2015,27(1):71-82.JI H Y,ZHU H Z.Progress of genome editing approaches towards gene therapy[J].Chinese Bulletin of Life Sciences,2015,27(1):71-82(in Chinese).
[60]LIANG Z,CHEN K L,ZHANG Y,et al.Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins[J].Nature Protocols,2018,13(3):413-430.
[61]GAO C X.The future of CRISPR technologies in a griculture[J].Nature Reviews Molecular Cell Biology,2018,19(5):1-2.
[2]张瑞成,李魏,潘素君,等.化学诱变在种质资源改良上的应用[J].分子植物育种,2017,15(12):5189-5196.ZHANG R C,LI W,PAN S J,et al.Application of chemical mutagenesis in improving germplasm resource[J].Molecular Plant Breeding,2017,15(12):5189-5196(in Chinese).
[3]MARTIN G B.Gene discovery for crop improvement[J].Current Opinion in Biotechnology,1998,9(2):220-226.
[4]HIEI Y,OHTA S,KOMARI T,et al.Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA[J].The Plant Journal,1994,6(2):271-282.
[5]KRYSAN P J,YOUNG J C,SUSSMAN M R.T-DNA as an insertional mutagen in Arabidopsis[J].The Plant Cell,1999,11(12):2283-2290.
[6]栾维江,孙宗修.Ac/Ds标签系统与水稻功能基因组学[J].植物生理与分子生物学学报,2005,31(5):441-450.LUAN W J,SUN Z X.Ac/Ds tagging system and functional genomics in rice[J].Journal of Plant Physiology and Molecular Biology,2005,31(5):441-450(in Chinese).
[7]HIROCHIKA H,SUGIMOTO K,OTSUKI Y,et al.Retrotransposons of rice involved in mutations induced by tissue culture[J].Proc Natl Acad Sci USA,1996,93(15):7783-7788.
[8]HIROCHIKA H.Contribution of the Tos17 retrotransposon to rice functional genomics[J].Current Opinion in Plant Biology,2001,4:118-122.
[9]PORTEUS M H,CARROLL D.Gene targeting using zinc finger nucleases[J].Nature Biotechnology,2005,23(8):967-973.
[10]PAVLETICH N P,PABO C O.Zinc Finger-DNA fecognition:Crystal structure of a Zif268-DNA complex at 2.1A[J].Science,1991,252(5007):809-817.
[11]CARROLL D.Progress and prospects:Zinc-finger nucleases as gene therapy agents[J].Gene Therapy,2008,15(22):1463-1468.
[12]BOLLER T,HE S Y.Innate immunity in plants:An arms race between pattern recognition receptors in plants and effectors in microbial pathogens[J].Science,2009,324(5928):742-744.
[13]KAY S,BONAS U.How Xanthomonas typeⅢeffectors manipulate the host plant[J].Current Opinion in Microbiology,2009,12(1):37-43.
[14]BOCH J,SCHOLZE H,SCHORNACK S,et al.Breaking the code of DNA binding specificity of TAL-typeⅢeffectors[J].Science,2009,326(5959):1509-1512.
[15]SANJANA N E,CONG L,ZHOU Y,et al.A transcription activatorlike effector toolbox for genome engineering[J].Nature Protocols,2012,7(1):171-192.
[16]ZHANG F,CONG L,LODATO S,et al.Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription[J].Nature Biotechnology,2011,29(2):149-154.
[17]MORBITZER R,R魻MER P,BOCH J et al.Regulation of selected genome loci using de novo-engineered transcription activator-like effector(TALE)-type transcription factors[J].Proc Natl Acad Sci USA,2010,107(50):21617-21622.
[18]CHRISTIAN M,CERMAK T,DOYLE E L et al.Targeting DNA doublestrand breaks with TAL effector nucleases[J].Genetics,2010,186(2):757-761.
[19]HOCKEMEYER D,WANG H,KIANI S,et al.Genetic engineering of human pluripotent cells using TALE nucleases[J].Nature Biotechnology,2011,29(8):731-734.
[20]WOOD A J,LO T,ZEITLER B,et al.Targeted genome editing across species using ZFNs and TALENs[J].Science,2011,333(6040):307.
[21]ZHANG Y,ZHANG F,LI X,et al.Transcription activator-like effector nucleases enable efficient plant genome engineering[J].Plant Physiology,2013,161(1):20-27.
[22]SHAN Q W,WANG Y P,CHEN K L,et al.Rapid and efficient gene modification in rice and Brachypodium using TALENs[J].Molecular Plant,2013,6(4):1365-1368.
[23]ISHINO Y,SHINAGAWA H,MAKINO K,et al.Nucleotide sequence of the iap gene,responsible for alkaline phosphatase isozyme conversion in Escherichia coli,and identification of the gene product[J].Journal of Bacteriology,1987,169(12):5429-5433.
[24]MOJICA F J M,JUEZ G,RODRIGUEZ-VALERA F.Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified Pstl sites[J].Molecular Microbiology,1993,9(3):613-621.
[25]MOJICA F J M,FERRER C,JUEZ G,et al.Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloforax mediterranei and Haloferax volcanii and could be involved in replicon partitioning[J].Molecular Microbiology,1995,17(1):85-93.
[26]MOJICA F J M,D魱EZ-VILLASE N′′OR C,SORIA E,et al.Biological significance of a family of regularly spaced repeats in the genomes of Archaea,Bacteria and mitochondria[J].Molecular Microbiology,2000,36(1):244-246.
[27]JANSEN R,EMBDEN J D A,GAASTRA W,et al.Identification of genes that are associated with DNA repeats in prokaryotes[J].Molecular Microbiology,2002,43(6):1565-1575.
[28]MOJICA F J M,D魱EZ-VILLASEN′′OR C,GARC魱A-MART魱NEZ J,et al.Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements[J].Journal of Molecular Evolution,2005,60(2):174-182.
[29]POURCEL C,SALVIGNOL G,VERGNAUD G.CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA,and provide additional tools for evolutionary studies[J].Microbiology,2005,151:653-663.
[30]BOLOTIN A,QUINQUIS B,SOROKIN A,et al.Clustered regularly interspaced short palindrome repeats(CRISPRs)have spacers of extrachromosomal origin[J].Microbiology,2005,151:2551-2561.
[31]MAKAROVA K S,ARAVIND L,GRISHIN N V,et al.A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis[J].Nucleic Acids Research,2002,30(2):482-496.
[32]MAKAROVA K S,GRISHIN N V,SHABALINA S A,et al.A putative RNA-interference-based immune system in prokaryotes:Computational analysis of the predicted enzymatic machinery,functional analogies with eukaryotic RNAi,and hypothetical mechanisms of action[J].Biology Direct,2006,1(7):1-26.
[33]BARRANGOU R,FREMAUX C,DEVEAU H,et al.CRISPR provides acquired resistance against viruses in prokaryotes[J].Science,2007,315(5819):1709-1712.
[34]MOJICA F J M,D魱EZ-VILLASEN′′OR C,GARCIA-MART魱NEZ J,et al.Short motif sequences determine the targets of the prokaryotic CRISPR defence system[J].Microbiology,2009,155:733-740.
[35]BROUNS S J J,JORE M M,LUNDGREN M,et al.Small CRISPRRNAs guide antiviral defense in prokaryotes[J].Science,2008,321(5891):960-964.
[36]CARTE J,WANG R Y,LI H,et al.Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes[J].Gens Development,2008,22(24):3489-3496.
[37]HAURWITZ R E,JINEK M,WIEDENHEFT B,et al.Sequence-and structure-specific RNA processing by a CRISPR endonuclease[J].Science,2010,329(5997):1355-1358.
[38]DELTCHEVA E,CHYLINSKI K,SHARMA C M,et al.CRISPR RNAmaturation by trans-encoded small RNA and host factor RNaseⅢ[J].Nature,2011,471(7340):602-607.
[39]GOTTESMAN S.Dicing defence in bacteria[J].Nature,2011,471(7340):588-589.
[40]MAKAROVA K S,Haft D H,BARRANGOU R,et al.Evolution and classification of the CRISPR-Cas systems[J].Nature Reviews Microbiology,2011,9(6):467-477.
[41]JINEK M,CHYLINSKI K,FONFARA I,et al,A programmable dualRNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,2012,337(6096):816-821.
[42]GASIUNAS G,BARRANGOU R,HORVATH P,et al.Cas9-crRNAribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J].Proc Natl Acad Sci USA,2012,109(39):2579-2586.
[43]CONG L,RAN F A,COX D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-823.
[44]MALI P,YANG L H,ESVELT K M,et al.RNA-guided human genome engineering via Cas9[J].Science,2013,339(6121):823-826.
[45]HWANG W Y,FU Y,REYON D,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system[J].Nature Biotechnology,2013,31(3):227-229.
[46]BASSETT A R,TIBBIT C,PONTING CP,et al.Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system[J].Cell Reports,2013,4(1):220-228.
[47]NAKAYAMA T,FISH M B,FISHER M,et al.Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis[J].Genesis,2013,51(12):835-843.
[48]NIU Y Y,SHEN B,CUI Y Q,et al.Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos[J].Cell,2014,156(4):836-843.
[49]SHAN Q W,WANG Y P,LI J,et al.Targeted genome modification of crop plants using a CRISPR-Cas system[J].Nature Biotechnology,2013,31(8):686-688.
[50]LI J F,NORVILLE J E,AACH J,et al.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9[J].Nature Biotechnology,2013,31(8):688-691.
[51]NEKRASOV V,STASKAWICZ B,WEIGEL D,et al.Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease[J].Nature Biotechnology,2013,31(8):691-693.
[52]MAO Y F,ZHANG H,XU N F,et al.Application of the CRISPR-Cas system for efficient genome engineering in plants[J].Molecular Plant,2013,6(6):2008-2011.
[53]FENG Z Y,ZHANG B T,DING W N,et al.Efficient genome editing in plants using a CRISPR/Cas system[J].Cell Research,2013,23(10):1229-1232.
[54]MIAO J,GUO D S,ZHANG J Z,et al.Targeted mutagenesis in rice using CRISPR-Cas system[J].Cell Research,2013,23(10):1233-1236.
[55]MA X L,ZHANG Q Y,ZHU Q L,et al.A robust CRISPR/Cas9 system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants[J].Molecular Plant,2015,8(8):1274-1284.
[56]ZETSCHE B,GOOTENBERG J S,ABUDAYYEH O O,et al.Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system[J].Cell,2015,163(3):759-771.
[57]HU J H,MILLER S M,GEURTS M H,et al.Evolved Cas9 variants with broad PAM compatibility and high DNA specificity[J].Nature,2018,556(7699):57-63.
[58]杨伟,李施施,张瑄,等.基于诱导多能干细胞的基因编辑和细胞治疗[J].中国细胞生物学学报,2015,37(1):90-99.YANG W,LI S S,ZHANG X,et al.Gene editing and cell therapy based on induced pluripotent stem cells[J].Chinese Journal of Cell Biology,2015,37(1):90-99(in Chinese).
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