非人灵长类动物基因编辑技术的应用及挑战
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摘要
建立有效的动物模型是研究人类疾病演进、开发新型治疗手段的重要方法。非人灵长类动物在进化发育、生理生化及病理方面和人类最接近,是研究人类疾病的理想动物模型。随着基因编辑技术的发展,研究者已经成功建立了多种模仿人类疾病的非人灵长类动物模型。但是CRISPR/Cas9的脱靶效应、嵌合突变以及基因敲入效率较低等突出问题也逐渐引起重视。本文综述了基因编辑技术在建立非人灵长类动物模型中的应用现状,提出了目前亟需解决的难点和应对策略,以期为高效、准确构建非人灵长类动物模型提供借鉴与参考。
There is a pressing urgency in the medical field to establish an effective animal model to study human diseases. Non-human primates,which are evolutionary,physiologically and pathologically closer to humans than any other animal,are thus considered to beexcellent animal models for studies of human diseases. With the development of gene manipulation technologies,researchers have successfullycreated a variety of non-human primate models via gene editing. Yet,there are still some challenges we have to face,such as off-target,mosaic mutants,and low efficiency of gene knock-in in CRISPR/Cas9. In this paper,we summarize the applications of gene editing in nonhuman primate models,as well as the existing problems and improvements,aimed to provide a reference for further research in non-human primate models mimicking human diseases.
There is a pressing urgency in the medical field to establish an effective animal model to study human diseases. Non-human primates,which are evolutionary,physiologically and pathologically closer to humans than any other animal,are thus considered to beexcellent animal models for studies of human diseases. With the development of gene manipulation technologies,researchers have successfullycreated a variety of non-human primate models via gene editing. Yet,there are still some challenges we have to face,such as off-target,mosaic mutants,and low efficiency of gene knock-in in CRISPR/Cas9. In this paper,we summarize the applications of gene editing in nonhuman primate models,as well as the existing problems and improvements,aimed to provide a reference for further research in non-human primate models mimicking human diseases.
引文
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[5]Chan AW,Chong KY,Martinovich C,et al.Transgenic monkeysproduced by retroviral gene transfer into mature oocytes[J].Science,2001,291(5502):309-312.
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[9]Sasaki E,Suemizu H,Shimada A,et al.Generation of transgenicnon-human primates with germline transmission[J].Nature,2009,459(7246):523-527.
[10]Niu Y,Yu Y,Bernat A,et al.Transgenic rhesus monkeys producedby gene transfer into early-cleavage-stage embryos using a simianimmunodeficiency virus-based vector[J].Proc Natl Acad Sci U S A,2010,107(41):17663-17667.
[11]Carroll D,Wahl LM.Genome engineering with zinc-fingernucleases[J].Genetics,2011,188(4):773-782.
[12]Cermak T,Doyle EL,Christian M,et al.Efficient design andassembly of custom TALEN and other TAL effector-basedconstructs for DNA targeting[J].Nucleic Acids Res,2011,39(12):e82.
[13]Liu H,Chen Y,Niu Y,et al.TALEN-mediated gene mutagenesis inrhesus and cynomolgus monkeys[J].Cell Stem Cell,2014,14(3):323-328.
[14]Chen Y,Yu J,Niu Y,et al.Modeling rett syndrome using TALENedited MECP2 mutant cynomolgus monkeys[J].Cell,2017,169(5):945-955.
[15]Ke Q,Li W,Lai X,et al.TALEN-based generation of a cynomolgusmonkey disease model for human microcephaly[J].Cell Res,2016,26(9):1048-1061.
[16]Horvath P and Barrangou R.CRISPR/Cas,the immune system ofbacteria and archaea[J].Science,2010,327(5962):167-170.
[17]Makarova KS,Wolf YI,Alkhnbashi OS,et al.An updatedevolutionary classification of CRISPR-Cas systems[J].Nat RevMicrobiol,2015,13(11):722-736.
[18]Abudayyeh OO,Gootenberg JS,Konermann S,et al.C2c2 is asingle-component programmable RNA-guided RNA-targetingCRISPR effector[J].Science,2016,353(6299):aaf5573.
[19]Zetsche B,Gootenberg JS,Abudayyeh OO,et al.Cpf1 is a singleRNA-guided endonuclease of a class 2 CRISPR-Cas system[J].Cell,2015,163(3):759-771.
[20]Fonfara I,Richter H,Bratovic M,et al.The CRISPR-associatedDNA-cleaving enzyme Cpf1 also processes precursor CRISPRRNA[J].Nature,2016,532(7600):517-521.
[21]Liu L,Li X,Wang J,et al.Two distant catalytic sites areresponsible for C2c2 RNase activities[J].Cell,2017,168(1-2):121-134.
[22]Cong L,Ran FA,Cox D,et al.Multiplex genome engineering usingCRISPR/Cas systems[J].Science,2013,339(6121):819-823.
[23]Mali P,Yang L,Esvelt KM,et al.RNA-guided human genomeengineering via Cas9[J].Science,2013,339(6121):823-826.
[24]Nishimasu H,Ran FA,Hsu PD,et al.Crystal structure of Cas9 incomplex with guide RNA and target DNA[J].Cell,2014,156(5):935-949.
[25]Niu Y,Shen B,Cui Y,et al.Generation of gene-modifiedcynomolgus monkey via Cas9/RNA-mediated gene targeting in onecell embryos[J].Cell,2014,156(4):836-843.
[26]Chen Y,Cui Y,Shen B,et al.Germline acquisition of Cas9/RNAmediated gene modifications in monkeys[J].Cell Res,2015,25(2):262-265.
[27]Chen Y,Zheng Y,Kang Y,et al.Functional disruption of thedystrophin gene in rhesus monkey using CRISPR/Cas9[J].HumMol Genet,2015,24(13):3764-3774.
[28]Kang Y,Zheng B,Shen B,et al.CRISPR/Cas9-mediated Dax1knockout in the monkey recapitulates human AHC-HH[J].HumMol Genet,2015,24(25):7255-7264.
[29]Wan H,Feng C,Teng F,et al.One-step generation of p53 genebiallelic mutant Cynomolgus monkey via the CRISPR/Cassystem[J].Cell Res,2015,25(2):258-261.
[30]Midic U,Hung PH,Vincent KA,et al.Quantitative assessment oftiming,efficiency,specificity and genetic mosaicism of CRISPR/Cas9-mediated gene editing of hemoglobin beta gene in rhesusmonkey embryos[J].Hum Mol Genet,2017,26(14):2678-2689.
[31]Rusk N.CRISPRs and epigenome editing[J].Nat Methods,2014,11(1):28.
[32]Fu Y,Foden JA,Khayter C,et al.High-frequency off-targetmutagenesis induced by CRISPR-Cas nucleases in humancells[J].Nat Biotechnol,2013,31(9):822-826.
[33]Mali P,Aach J,Stranges PB,et al.CAS9 transcriptional activatorsfor target specificity screening and paired nickases for cooperativegenome take engineering[J].Nat Biotechnol,2013,31(9):833-838.
[34]Pattanayak V,Lin S,Guilinger JP,et al.High-throughput profilingof off-target DNA cleavage reveals RNA-programmed Cas9nuclease specificity[J].Nat Biotechnol,2013,31(9):839-843.
[35]Liang P,Xu Y,Zhang X,et al.CRISPR/Cas9-mediated geneediting in human tripronuclear zygotes[J].Protein Cell,2015,6(5):363-372.
[36]Ran FA,Hsu PD,Lin CY,et al.Double nicking by RNA-guidedCRISPR Cas9 for enhanced genome editing specificity[J].Cell,2013,154(6):1380-1389.
[37]Guilinger JP,Thompson DB and Liu DR.Fusion of catalyticallyinactive Cas9 to Fok I nuclease improves the specificity of genomemodification[J].Nat Biotechnol,2014,32(6):577-582.
[38]Shengdar QT,Nicolas W,Cyd K,et al.Dimeric CRISPR RNAguided Fok I nucleases for highly specific genome editing[J].NatBiotechnol,2014,32(6):569-576.
[39]Slaymaker IM,Gao L,Zetsche B,et al.Rationally engineered Cas9nucleases with improved specificity[J].Science,2016,351(6268):84-88.
[40]Kleinstiver BP,Pattanayak V,Prew MS,et al.High-fidelityCRISPR-Cas9 nucleases with no detectable genome-wide off-targeteffects[J].Nature,2016,529(7587):490-495.
[41]Liu KI,Ramli MN,Woo CW,et al.A chemical-inducible CRISPRCas9 system for rapid control of genome editing[J].Nat ChemBiol,2016,12(11):980-987.
[42]Nihongaki Y,Kawano F,Nakajima TP,et al.PhotoactivatableCRISPR-Cas9 for optogenetic genome editing[J].NatBiotechnol,2015,33(7):755-760.
[43]Rauch BJ,Silvis MR,Hultquist JF,et al.Inhibition of CRISPRCas9 with Bacteriophage Proteins[J].Cell,2017,168(1-2):150-158.
[44]Lee CM,Cradick TJ,Fine EJ,et al.Nuclease target site selectionfor maximizing on-target activity and minimizing off-target effects ingenome editing[J].Mol Ther,2016,24(3):475-487.
[45]Rastogi A,Murik O,Bowler C,et al.Phyto CRISP-Ex:a web-basedand stand-alone application to find specific target sequences forCRISPR/CAS editing[J].BMC Bioinformatics,2016,17(1):261.
[46]Hsu PD,Scott DA,Weinstein JA,et al.DNA targeting specificity ofRNA-guided Cas9 nucleases[J].Nat Biotechnol,2013,31(9):827-832.
[47]Seung WC,Sojung K,Yongsub K,et al.Analysis of off-targeteffects of CRISPR/Cas-derived RNA-guided endonucleases andnickases[J].Genome Res,2014,24(1):132-141.
[48]Fu Y,Sander JD,Reyon D,et al.Improving CRISPR-Cas nucleasespecificity using truncated guide RNAs[J].Nat Biotechnol,2014,32(3):279-284.
[49]Wyvekens N,Topkar VV,Khayter C,et al.Dimeric CRISPR RNAguided Fok I-d Cas9 nucleases directed by truncated g RNAs forhighly specific genome editing[J].Hum Gene Ther,2015,26(7):425-431.
[50]Tu Z,Yang W,Yan S,et al.Promoting Cas9 degradation reducesmosaic mutations in non-human primate embryos[J].Sci Rep,2017,7(1):42081.
[51]Hashimoto M,Yamashita Y,Takemoto T.Electroporation of Cas9protein/sg RNA into early pronuclear zygotes generates non-mosaicmutants in the mouse[J].Dev Biol,2016,418(1):1-9.
[52]Platt RJ,Chen S,Zhou Y,et al.CRISPR-Cas9 knockin mice forgenome editing and cancer modeling[J].Cell,2014,159(2):440-455.
[53]Sakurai T,Watanabe S,Kamiyoshi A,et al.A single blastocystassay optimized for detecting CRISPR/Cas9 system-induced indelmutations in mice[J].BMC Biotechnol,2014,14(1):69.
[54]Tsai SQ,Zheng Z,Nguyen NT,et al.GUIDE-seq enablesgenome-wide profiling of off-target cleavage by CRISPR-Casnucleases[J].Nat Biotechnol,2015,33(2):187-197.
[55]Kim D,Bae S,Park J,et al.Digenome-seq:genome-wide profilingof CRISPR-Cas9 off-target effects in human cells[J].NatMethods,2015,12(3):237-243.
[56]Maruyama T,Dougan SK,Truttmann MC,et al.Increasing theefficiency of precise genome editing with CRISPR-Cas9 byinhibition of nonhomologous end joining[J].Nat Biotechnol,2015,33(5):538-542.
[57]Aida T,Chiyo K,Usami T,et al.Cloning-free CRISPR/Cas systemfacilitates functional cassette knock-in in mice[J].Genome Biol,2015,16(1):87.
[58]David Cyranoski.CRISPR gene-editing tested in a person for thefirst time[J].Nature,2016,539(7630):479.
[59]Ma H,Marti-Gutierrez N,Park SW,et al.Correction of a pathogenicgene mutation in human embryos[J].Nature,2017,548(7668):413-419.
[60]Wu Y,Liang D,Wang Y,et al.Correction of a genetic disease inmouse via use of CRISPR-Cas9[J].Cell Stem Cell,2013,13(6):659-662.
[61]Liu Y,Qi X,Zeng Z,et al.CRISPR/Cas9-mediated p53 and ptendual mutation accelerates he patocarcinogenesis in adult hepatitisB virus transgenic mice[J].Sci Rep,2017,7(1):2796.
[62]Reardon S.Leukaemia success heralds wave of gene-editingtherapies[J].Nature,2015,12;527(7577):146-147.
[63]Wang Z,Pan Q,Gendron P,et al.CRISPR/Cas9-derived mutationsboth inhibit HIV-1 replication and accelerate viral escape[J].Cell Rep,2016,15(3):481-489.
[2]Hoke A,Ray M.Rodent models of chemotherapy-induced peripheralneuropathy[J].ILAR J,2014,54(3):273-281.
[3]Yan G,Zhang G,Fang X,et al.Genome sequencing and comparisonof two nonhuman primate animal models,the cynomolgus andChinese rhesus macaques[J].Nat Biotechnol,2011,29(11):1019-1023.
[4]Gibbs RA,Rogers J,Katze MG,et al.Evolutionary and biomedicalinsights from the rhesus macaque genome[J].Science,2007,316(5822):222-234.
[5]Chan AW,Chong KY,Martinovich C,et al.Transgenic monkeysproduced by retroviral gene transfer into mature oocytes[J].Science,2001,291(5502):309-312.
[6]Yang SH,Cheng PH,Banta H,et al.Towards a transgenic model ofHuntington’s disease in a non-human primate[J].Nature,2008,453(7197):921-924.
[7]Niu Y,Guo X,Chen Y,et al.Early Parkinson's disease symptoms inα-synuclein transgenic monkeys[J].Hum Mol Genet,2015,24(8):2308-2317.
[8]Liu Z,Li X,Zhang JT,et al.Autism-like behaviours and germlinetransmission in transgenic monkeys overexpressing Me CP2[J].Nature,2016,530(7588):98-102.
[9]Sasaki E,Suemizu H,Shimada A,et al.Generation of transgenicnon-human primates with germline transmission[J].Nature,2009,459(7246):523-527.
[10]Niu Y,Yu Y,Bernat A,et al.Transgenic rhesus monkeys producedby gene transfer into early-cleavage-stage embryos using a simianimmunodeficiency virus-based vector[J].Proc Natl Acad Sci U S A,2010,107(41):17663-17667.
[11]Carroll D,Wahl LM.Genome engineering with zinc-fingernucleases[J].Genetics,2011,188(4):773-782.
[12]Cermak T,Doyle EL,Christian M,et al.Efficient design andassembly of custom TALEN and other TAL effector-basedconstructs for DNA targeting[J].Nucleic Acids Res,2011,39(12):e82.
[13]Liu H,Chen Y,Niu Y,et al.TALEN-mediated gene mutagenesis inrhesus and cynomolgus monkeys[J].Cell Stem Cell,2014,14(3):323-328.
[14]Chen Y,Yu J,Niu Y,et al.Modeling rett syndrome using TALENedited MECP2 mutant cynomolgus monkeys[J].Cell,2017,169(5):945-955.
[15]Ke Q,Li W,Lai X,et al.TALEN-based generation of a cynomolgusmonkey disease model for human microcephaly[J].Cell Res,2016,26(9):1048-1061.
[16]Horvath P and Barrangou R.CRISPR/Cas,the immune system ofbacteria and archaea[J].Science,2010,327(5962):167-170.
[17]Makarova KS,Wolf YI,Alkhnbashi OS,et al.An updatedevolutionary classification of CRISPR-Cas systems[J].Nat RevMicrobiol,2015,13(11):722-736.
[18]Abudayyeh OO,Gootenberg JS,Konermann S,et al.C2c2 is asingle-component programmable RNA-guided RNA-targetingCRISPR effector[J].Science,2016,353(6299):aaf5573.
[19]Zetsche B,Gootenberg JS,Abudayyeh OO,et al.Cpf1 is a singleRNA-guided endonuclease of a class 2 CRISPR-Cas system[J].Cell,2015,163(3):759-771.
[20]Fonfara I,Richter H,Bratovic M,et al.The CRISPR-associatedDNA-cleaving enzyme Cpf1 also processes precursor CRISPRRNA[J].Nature,2016,532(7600):517-521.
[21]Liu L,Li X,Wang J,et al.Two distant catalytic sites areresponsible for C2c2 RNase activities[J].Cell,2017,168(1-2):121-134.
[22]Cong L,Ran FA,Cox D,et al.Multiplex genome engineering usingCRISPR/Cas systems[J].Science,2013,339(6121):819-823.
[23]Mali P,Yang L,Esvelt KM,et al.RNA-guided human genomeengineering via Cas9[J].Science,2013,339(6121):823-826.
[24]Nishimasu H,Ran FA,Hsu PD,et al.Crystal structure of Cas9 incomplex with guide RNA and target DNA[J].Cell,2014,156(5):935-949.
[25]Niu Y,Shen B,Cui Y,et al.Generation of gene-modifiedcynomolgus monkey via Cas9/RNA-mediated gene targeting in onecell embryos[J].Cell,2014,156(4):836-843.
[26]Chen Y,Cui Y,Shen B,et al.Germline acquisition of Cas9/RNAmediated gene modifications in monkeys[J].Cell Res,2015,25(2):262-265.
[27]Chen Y,Zheng Y,Kang Y,et al.Functional disruption of thedystrophin gene in rhesus monkey using CRISPR/Cas9[J].HumMol Genet,2015,24(13):3764-3774.
[28]Kang Y,Zheng B,Shen B,et al.CRISPR/Cas9-mediated Dax1knockout in the monkey recapitulates human AHC-HH[J].HumMol Genet,2015,24(25):7255-7264.
[29]Wan H,Feng C,Teng F,et al.One-step generation of p53 genebiallelic mutant Cynomolgus monkey via the CRISPR/Cassystem[J].Cell Res,2015,25(2):258-261.
[30]Midic U,Hung PH,Vincent KA,et al.Quantitative assessment oftiming,efficiency,specificity and genetic mosaicism of CRISPR/Cas9-mediated gene editing of hemoglobin beta gene in rhesusmonkey embryos[J].Hum Mol Genet,2017,26(14):2678-2689.
[31]Rusk N.CRISPRs and epigenome editing[J].Nat Methods,2014,11(1):28.
[32]Fu Y,Foden JA,Khayter C,et al.High-frequency off-targetmutagenesis induced by CRISPR-Cas nucleases in humancells[J].Nat Biotechnol,2013,31(9):822-826.
[33]Mali P,Aach J,Stranges PB,et al.CAS9 transcriptional activatorsfor target specificity screening and paired nickases for cooperativegenome take engineering[J].Nat Biotechnol,2013,31(9):833-838.
[34]Pattanayak V,Lin S,Guilinger JP,et al.High-throughput profilingof off-target DNA cleavage reveals RNA-programmed Cas9nuclease specificity[J].Nat Biotechnol,2013,31(9):839-843.
[35]Liang P,Xu Y,Zhang X,et al.CRISPR/Cas9-mediated geneediting in human tripronuclear zygotes[J].Protein Cell,2015,6(5):363-372.
[36]Ran FA,Hsu PD,Lin CY,et al.Double nicking by RNA-guidedCRISPR Cas9 for enhanced genome editing specificity[J].Cell,2013,154(6):1380-1389.
[37]Guilinger JP,Thompson DB and Liu DR.Fusion of catalyticallyinactive Cas9 to Fok I nuclease improves the specificity of genomemodification[J].Nat Biotechnol,2014,32(6):577-582.
[38]Shengdar QT,Nicolas W,Cyd K,et al.Dimeric CRISPR RNAguided Fok I nucleases for highly specific genome editing[J].NatBiotechnol,2014,32(6):569-576.
[39]Slaymaker IM,Gao L,Zetsche B,et al.Rationally engineered Cas9nucleases with improved specificity[J].Science,2016,351(6268):84-88.
[40]Kleinstiver BP,Pattanayak V,Prew MS,et al.High-fidelityCRISPR-Cas9 nucleases with no detectable genome-wide off-targeteffects[J].Nature,2016,529(7587):490-495.
[41]Liu KI,Ramli MN,Woo CW,et al.A chemical-inducible CRISPRCas9 system for rapid control of genome editing[J].Nat ChemBiol,2016,12(11):980-987.
[42]Nihongaki Y,Kawano F,Nakajima TP,et al.PhotoactivatableCRISPR-Cas9 for optogenetic genome editing[J].NatBiotechnol,2015,33(7):755-760.
[43]Rauch BJ,Silvis MR,Hultquist JF,et al.Inhibition of CRISPRCas9 with Bacteriophage Proteins[J].Cell,2017,168(1-2):150-158.
[44]Lee CM,Cradick TJ,Fine EJ,et al.Nuclease target site selectionfor maximizing on-target activity and minimizing off-target effects ingenome editing[J].Mol Ther,2016,24(3):475-487.
[45]Rastogi A,Murik O,Bowler C,et al.Phyto CRISP-Ex:a web-basedand stand-alone application to find specific target sequences forCRISPR/CAS editing[J].BMC Bioinformatics,2016,17(1):261.
[46]Hsu PD,Scott DA,Weinstein JA,et al.DNA targeting specificity ofRNA-guided Cas9 nucleases[J].Nat Biotechnol,2013,31(9):827-832.
[47]Seung WC,Sojung K,Yongsub K,et al.Analysis of off-targeteffects of CRISPR/Cas-derived RNA-guided endonucleases andnickases[J].Genome Res,2014,24(1):132-141.
[48]Fu Y,Sander JD,Reyon D,et al.Improving CRISPR-Cas nucleasespecificity using truncated guide RNAs[J].Nat Biotechnol,2014,32(3):279-284.
[49]Wyvekens N,Topkar VV,Khayter C,et al.Dimeric CRISPR RNAguided Fok I-d Cas9 nucleases directed by truncated g RNAs forhighly specific genome editing[J].Hum Gene Ther,2015,26(7):425-431.
[50]Tu Z,Yang W,Yan S,et al.Promoting Cas9 degradation reducesmosaic mutations in non-human primate embryos[J].Sci Rep,2017,7(1):42081.
[51]Hashimoto M,Yamashita Y,Takemoto T.Electroporation of Cas9protein/sg RNA into early pronuclear zygotes generates non-mosaicmutants in the mouse[J].Dev Biol,2016,418(1):1-9.
[52]Platt RJ,Chen S,Zhou Y,et al.CRISPR-Cas9 knockin mice forgenome editing and cancer modeling[J].Cell,2014,159(2):440-455.
[53]Sakurai T,Watanabe S,Kamiyoshi A,et al.A single blastocystassay optimized for detecting CRISPR/Cas9 system-induced indelmutations in mice[J].BMC Biotechnol,2014,14(1):69.
[54]Tsai SQ,Zheng Z,Nguyen NT,et al.GUIDE-seq enablesgenome-wide profiling of off-target cleavage by CRISPR-Casnucleases[J].Nat Biotechnol,2015,33(2):187-197.
[55]Kim D,Bae S,Park J,et al.Digenome-seq:genome-wide profilingof CRISPR-Cas9 off-target effects in human cells[J].NatMethods,2015,12(3):237-243.
[56]Maruyama T,Dougan SK,Truttmann MC,et al.Increasing theefficiency of precise genome editing with CRISPR-Cas9 byinhibition of nonhomologous end joining[J].Nat Biotechnol,2015,33(5):538-542.
[57]Aida T,Chiyo K,Usami T,et al.Cloning-free CRISPR/Cas systemfacilitates functional cassette knock-in in mice[J].Genome Biol,2015,16(1):87.
[58]David Cyranoski.CRISPR gene-editing tested in a person for thefirst time[J].Nature,2016,539(7630):479.
[59]Ma H,Marti-Gutierrez N,Park SW,et al.Correction of a pathogenicgene mutation in human embryos[J].Nature,2017,548(7668):413-419.
[60]Wu Y,Liang D,Wang Y,et al.Correction of a genetic disease inmouse via use of CRISPR-Cas9[J].Cell Stem Cell,2013,13(6):659-662.
[61]Liu Y,Qi X,Zeng Z,et al.CRISPR/Cas9-mediated p53 and ptendual mutation accelerates he patocarcinogenesis in adult hepatitisB virus transgenic mice[J].Sci Rep,2017,7(1):2796.
[62]Reardon S.Leukaemia success heralds wave of gene-editingtherapies[J].Nature,2015,12;527(7577):146-147.
[63]Wang Z,Pan Q,Gendron P,et al.CRISPR/Cas9-derived mutationsboth inhibit HIV-1 replication and accelerate viral escape[J].Cell Rep,2016,15(3):481-489.