CRISPR/Cas9技术在非模式植物中的应用进展
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摘要
基因组编辑技术的出现对植物遗传育种及作物性状的改良产生了深远意义。CRISPR/Cas(clustered regularly interspaced short palindromic repeat)是由成簇规律间隔短回文重复序列及其关联蛋白组成的免疫系统,其作用是原核生物(40%细菌和90%古细菌)用来抵抗外源遗传物质(噬菌体和病毒)的入侵。该技术实现了对基因组中多个靶基因同时进行编辑,与前两代基因编辑技术:锌指核酶(ZFNs)和转录激活因子样效应物核酶(TALENs)相比更加简单、廉价、高效。目前CRISPR/Cas9基因编辑技术已在拟南芥(Arabidopsis thaliana)、烟草(Nicotiana benthamiana)、水稻(Oryza sativa)、小麦(Triticum aestivum)、玉米(Zea mays)、番茄(tomato)等模式植物和多数大作物中实现了定点基因组编辑,其应用范围不断地向各类植物扩展。但与模式植物和一些大作物相比,CRISPR/Cas9基因编辑技术在非模式植物,尤其在一些小作物的应用中存在如载体构建、靶点设计、脱靶检测、同源重组等问题有待进一步完善。该文对CRISPR/Cas9技术在非模式植物与小作物研究的最新研究进展进行了总结,讨论了该技术目前在非模式植物、小作物应用的局限性,在此基础上提出了相关改进策略,并对CRISPR/Cas9系统在非模式植物中的研究前景进行了展望。
The emergence of genome editing technology has far-reaching significance for plant genetic breeding and improvement of crop traits. CRISPR/Cas( clustered ordered interspaced short palindromic repeat) is a cluster of regularly spaced short palindromic repeats used by prokaryotes( 40% bacteria and 90% archaea) to resist the invasion of foreign genetic material( phage and viruses). The immune system consists of its associated proteins. CRISPR acts as an RNAbased acquired immune defense system. Its spacer sequence shares homology with phage or plasmid sequences and can use target-specific RNA to direct the Cas protein to target genes that are genetically incorrect in almost all organisms and cells. Compared with the gene editing technology of zinc finger nuclease ZFN and transcriptional activator-like effector ribozyme TALEN,it has the advantages of being efficient,cheap,and easy to operate,rapidly surpassing the previous technology,and becoming the hottest site-specific gene editing tool. The CRISPR/Cas9 gene editing technology has been successfully implemented in Arabidopsis thaliana,Nicotiana abenthamiana,Oryza sativa,Triticum aestivum,Zea mays,tomato and other large-scale plants. The application of fixed-point genome editing also extends to various types of plants. However,CRISPR/Cas9 gene editing technology has low application in non-patterns,especially in some small crops,compared with model species and some large crops. The problems such as vector construction,target design,offtarget detection,and homologous recombination need to be further addressed. This paper summarizes recent advances in CRISPR/Cas9 technology and research on non-model plants and small crops,and discusses the limitations of this technology in the application of non-model plants and small crops. Finally,the research prospects of the CRISPR/Cas9 system were prospected,which provided references for related researchers. It is believed that with the further development of CRISPR/Cas9 technology,these problems will eventually be overcome,and its emergence will certainly bring about better development of plant genetic engineering.
The emergence of genome editing technology has far-reaching significance for plant genetic breeding and improvement of crop traits. CRISPR/Cas( clustered ordered interspaced short palindromic repeat) is a cluster of regularly spaced short palindromic repeats used by prokaryotes( 40% bacteria and 90% archaea) to resist the invasion of foreign genetic material( phage and viruses). The immune system consists of its associated proteins. CRISPR acts as an RNAbased acquired immune defense system. Its spacer sequence shares homology with phage or plasmid sequences and can use target-specific RNA to direct the Cas protein to target genes that are genetically incorrect in almost all organisms and cells. Compared with the gene editing technology of zinc finger nuclease ZFN and transcriptional activator-like effector ribozyme TALEN,it has the advantages of being efficient,cheap,and easy to operate,rapidly surpassing the previous technology,and becoming the hottest site-specific gene editing tool. The CRISPR/Cas9 gene editing technology has been successfully implemented in Arabidopsis thaliana,Nicotiana abenthamiana,Oryza sativa,Triticum aestivum,Zea mays,tomato and other large-scale plants. The application of fixed-point genome editing also extends to various types of plants. However,CRISPR/Cas9 gene editing technology has low application in non-patterns,especially in some small crops,compared with model species and some large crops. The problems such as vector construction,target design,offtarget detection,and homologous recombination need to be further addressed. This paper summarizes recent advances in CRISPR/Cas9 technology and research on non-model plants and small crops,and discusses the limitations of this technology in the application of non-model plants and small crops. Finally,the research prospects of the CRISPR/Cas9 system were prospected,which provided references for related researchers. It is believed that with the further development of CRISPR/Cas9 technology,these problems will eventually be overcome,and its emergence will certainly bring about better development of plant genetic engineering.
引文
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ELORRIAGA E,2016.Asexual gene drive in populus?Results from CRISPR/Cas9 mutagenesis of floral genes for genetic containment[C].PAG XXVI,San Diego,CA.
EYQUEM J,MANSILLA-SOTO J,GIAVRIDIS T,et al.,2017.Targeting a CAR to the TRAC locus with CRISPR/Cas9enhancestumour rejection[J].Nature,543(7643):113.
FAUSER F,SCHIML S,PUCHTA H,2014.Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana[J].Plant J,79(2):348-359.
HENDEL A,KILDEBECK EJ,Fine EJ,et al.,2014.Quantifying genome-editing outcomes at endogenous loci with SMRTsequencing[J].Cell Rep,7(1):293.
HU CH,DENG GM,SUN XX,et al.,2017.Establishment of an efficient CRISPR/Cas9-mediated gene editing system in banana[J].Sci Agric Sin,50(7):1294-1301.[胡春华,邓贵明,孙晓玄,等,2017.香蕉CRISPR/Cas9基因编辑技术体系的建立[J].中国农业科学,50(7):1294-1301.]
JIA H,WANG N,2014.Targeted genome editing of sweet orange using Cas9/sgRNA[J].PLoS ONE,9(4):e93806.
KHATODIA S,BHATOTIA K,TUTEJA N,2017.Development of CRISPR/Cas9 mediated virus resistance in agriculturally important crops[J].Bioengineered,8(3):274-279.
KIM D,KIM J,HUR JK,et al.,2016.Genome-wide analysis reveals specificities of Cpf1 endo nucleases in human cells[J].Nat Biotechnol,34(8):863-868.
KLEINSTIVER BP,PATTANAYAK V,PREW MS,et al.,2016.High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects[J].Nature,529(7587):490-495.
LANDER ES,2016.The heroes of CRISPR[J].Cell,164(2):18-28.
LEI Y,LU L,LIU HY,et al.,2014.CRISPR-P:A web tool for synthetic single-guide RNA design of CRISPR-system in plants[J].Mol Plant,7(9):1494-1496.
LOWDER LG,ZHANG D,BALTES NJ,et al.,2015.ACRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation[J].Plant Physiol,169(2):971-985.
MENG Y,HOU Y,WANG H,et al.,2017.Targeted mutagenesis by CRISPR/Cas9 system in the model legume Medicago truncatula[J].Plant Cell Rep,36(2):371-374.
MIKAMI M,TOKI S,ENDO M,2015.Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice[J].Plant Mol Biol,88(6):561-572.
OSAKABE Y,OSAKABE K,2015.Genome editing with engineered nucleases in plants[J].Plant Cell Physiol,56(3):389-400.
PAN C,YE L,QIN L,et al.,2016.CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations[J].Sci Rep,78(6):46916.
PETOLINO JF,2015.Genome editing in plants via designed zinc finger nucleases[J].In Vitro Cell Dev Biol Plant,51(1):1-8.
PUCHTA H,FAUSER F,2014.Synthetic nucleases for genome engineering in plants:Prospects for a bright future[J].Plant J,78(5):727-741.
PUCHTA H,2017.Applying CRISPR/Cas for genome engineering in plants:the best is yet to come[J].Curr Opin Plant Biol,25(36):1-8.
RAN FA,HSU PD,WRIGHT J,et al.,2013.Genome engineering using the CRISPR-Cas9 system[J].Nat Protoc,8(11):2281-2308.
REN C,LIU XJ,ZHANG Z,et al.,2016.CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay(Vitis vinifera L.)[J].Sci Rep,(6):32289.
SAMANTA MK,DEY A,GAYEN S,2016.CRISPR/Cas9:An advanced tool for editing plant genomes[J].Transg Res,25(5):561-573.
SANJANA NE,CONG L,ZHOU Y,et al.,2012.A transcription activator-like effector toolbox for genome engineering[J].Nat Protoc,7(1):171-192.
SHEN B,ZHANG J,WU HY,et al.,2013.Generation of gene-modified mice via Cas9/RNA-mediated gene targeting[J].Cell Res,23(5):720-723.
SHEN B,ZHANG W,ZHANG J,et al.,2014.Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects[J].Nat Methods,11(4):399-402.
SUGANO SS,SHIRAKAWA M,TAKAGI JT,et al.,2014.CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L.[J].Plant Cell Physiol,55(3):475-481.
SUN X,HU Z,CHEN R,et al.,2015.Targeted mutagenesis in soybean using the CRISPR-Cas9 system[J].Sci Rep,(5):10342.
SUN YG,ZHANG X,WU CY,et al.,2016.Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase[J].Mol Plant,9(4):628-631.
SVITASHEV S,YOUNG JK,SCHWARTZ C,et al.,2015.Targeted mutagenesis,precise gene editing,and site-specific gene insertion in maize using Cas9 and guide RNA[J].Plant Physiol,169(2):931-945.
UNCKLESS RL,CLARK AG,MESSER PW,2017.Evolution of resistance against CRISPR/Cas9 gene drive[J].Genetics,205(2):827.
VAN ZEIJL A,WARDHANI TAK,SEIFI KALHOR M,et al.,2018.CRISPR/Cas9-mediated mutagenesis of four putative symbiosis genes of the tropical tree Parasponia andersonii reveals novel phenotypes[J].Front Plant Sci,(9):284.
WANG LX,WANG LL,TAN Q,et al.,2016.Efficient inactivation of symbiotic nitrogen fixation related genes in lotus japonicas using CRISPR-Cas9[J].Front Plant Sci,7(703).
WANG MG,LU YM,BOTELLA JR,et al.,2017.Gene targeting by homology-directed repair in rice using ageminivirusbased CRISPR/Cas9 system[J].Mol Plant,10(7):1007-1010.
WANG S,ZHANG S,WANG W,et al.,2015.Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system[J].Plant Cell Rep,1473-1476(9):10342.
WEI Y,ZHANG XH,LI DL,2017.The“new favorite”of gene editing technology-single base editors[J].Hereditas,39(12):1115-1121.[魏瑜,张晓辉,李大力,2017.基因编辑之“新宠”-单碱基基因组编辑系统[J].遗传,39(12):1115-1121.]
XIE K,MINKENBERG B,YANG Y,2015.Boosting CRISPR/Cas9 multiplex editing capability with the endogenoust RNA-processing system[J].Proc Natl Acad Sci USA,112(11):3570-3575.
YANG H,WANG HY,SHIVALILA CS,et al.,2013.Onestep generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering[J].Cell,154(6):1370-1379.
YIN KQ,HAN T,LIU G,et al.,2015.Ageminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing[J].Sci Rep,7(5):14926.
ZHANG B,YANG X,YANG C,et al.,2016a.Exploiting the CRISPR/Cas9 system for targeted genome mutagenesis in Petunia[J].Sci Rep,(6):20315.
ZHANG D,LI Z,LI JF,2016b.Targeted gene manipulation in plants using the CRISPR/Cas technology[J].J Genet Genom,43(5):251-262.
ZHANG L,ZHOU Q,2014.CRISPR/Cas technology:A revolutionary approach for genome engineering[J].Sci Chin Life Sci,57(6):639-640.
BOCH J,BONAS U,2010.Xanthomonas AvrBs3 family-type III effectors:Discovery and function[J].Ann Rev Phytopathol,48:419-436.
CENCIC R,MIURA H,MALINA A,et al.,2014.Protospacer adjacent motif(PAM)-distal sequences engage CRISPR Cas9DNA target cleavage[J].PLoS ONE,9(10):e109213.
CRADICK TJ,ANTICO CJ,BAO G,2014.High-throughput cellular screening of engineered nuclease activity using the single-strand annealing assay andluciferase reporter[J].Methods Mol Biol,(1114):339-352.
DEMIRCI Y,ZHANG B,UNVER T,2018.CRISPR/Cas9:An RNA-guided highly precise synthetic tool for plant genome editing[J].Cell Physiol,233(3):32-36.
D'HALLUIN K,RUITER R,2013.Directed genome engineering for genome optimization[J].Int J Dev Biol,57(6-8):621-627.
DOUDNA JA,CHARPENTIER E,2014.Genome editing:The new frontier of genome engineering with CRISPR-Cas9[J].Science,346(6213):1258096.
ELORRIAGA E,2016.Asexual gene drive in populus?Results from CRISPR/Cas9 mutagenesis of floral genes for genetic containment[C].PAG XXVI,San Diego,CA.
EYQUEM J,MANSILLA-SOTO J,GIAVRIDIS T,et al.,2017.Targeting a CAR to the TRAC locus with CRISPR/Cas9enhancestumour rejection[J].Nature,543(7643):113.
FAUSER F,SCHIML S,PUCHTA H,2014.Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana[J].Plant J,79(2):348-359.
HENDEL A,KILDEBECK EJ,Fine EJ,et al.,2014.Quantifying genome-editing outcomes at endogenous loci with SMRTsequencing[J].Cell Rep,7(1):293.
HU CH,DENG GM,SUN XX,et al.,2017.Establishment of an efficient CRISPR/Cas9-mediated gene editing system in banana[J].Sci Agric Sin,50(7):1294-1301.[胡春华,邓贵明,孙晓玄,等,2017.香蕉CRISPR/Cas9基因编辑技术体系的建立[J].中国农业科学,50(7):1294-1301.]
JIA H,WANG N,2014.Targeted genome editing of sweet orange using Cas9/sgRNA[J].PLoS ONE,9(4):e93806.
KHATODIA S,BHATOTIA K,TUTEJA N,2017.Development of CRISPR/Cas9 mediated virus resistance in agriculturally important crops[J].Bioengineered,8(3):274-279.
KIM D,KIM J,HUR JK,et al.,2016.Genome-wide analysis reveals specificities of Cpf1 endo nucleases in human cells[J].Nat Biotechnol,34(8):863-868.
KLEINSTIVER BP,PATTANAYAK V,PREW MS,et al.,2016.High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects[J].Nature,529(7587):490-495.
LANDER ES,2016.The heroes of CRISPR[J].Cell,164(2):18-28.
LEI Y,LU L,LIU HY,et al.,2014.CRISPR-P:A web tool for synthetic single-guide RNA design of CRISPR-system in plants[J].Mol Plant,7(9):1494-1496.
LOWDER LG,ZHANG D,BALTES NJ,et al.,2015.ACRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation[J].Plant Physiol,169(2):971-985.
MENG Y,HOU Y,WANG H,et al.,2017.Targeted mutagenesis by CRISPR/Cas9 system in the model legume Medicago truncatula[J].Plant Cell Rep,36(2):371-374.
MIKAMI M,TOKI S,ENDO M,2015.Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice[J].Plant Mol Biol,88(6):561-572.
OSAKABE Y,OSAKABE K,2015.Genome editing with engineered nucleases in plants[J].Plant Cell Physiol,56(3):389-400.
PAN C,YE L,QIN L,et al.,2016.CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations[J].Sci Rep,78(6):46916.
PETOLINO JF,2015.Genome editing in plants via designed zinc finger nucleases[J].In Vitro Cell Dev Biol Plant,51(1):1-8.
PUCHTA H,FAUSER F,2014.Synthetic nucleases for genome engineering in plants:Prospects for a bright future[J].Plant J,78(5):727-741.
PUCHTA H,2017.Applying CRISPR/Cas for genome engineering in plants:the best is yet to come[J].Curr Opin Plant Biol,25(36):1-8.
RAN FA,HSU PD,WRIGHT J,et al.,2013.Genome engineering using the CRISPR-Cas9 system[J].Nat Protoc,8(11):2281-2308.
REN C,LIU XJ,ZHANG Z,et al.,2016.CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay(Vitis vinifera L.)[J].Sci Rep,(6):32289.
SAMANTA MK,DEY A,GAYEN S,2016.CRISPR/Cas9:An advanced tool for editing plant genomes[J].Transg Res,25(5):561-573.
SANJANA NE,CONG L,ZHOU Y,et al.,2012.A transcription activator-like effector toolbox for genome engineering[J].Nat Protoc,7(1):171-192.
SHEN B,ZHANG J,WU HY,et al.,2013.Generation of gene-modified mice via Cas9/RNA-mediated gene targeting[J].Cell Res,23(5):720-723.
SHEN B,ZHANG W,ZHANG J,et al.,2014.Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects[J].Nat Methods,11(4):399-402.
SUGANO SS,SHIRAKAWA M,TAKAGI JT,et al.,2014.CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L.[J].Plant Cell Physiol,55(3):475-481.
SUN X,HU Z,CHEN R,et al.,2015.Targeted mutagenesis in soybean using the CRISPR-Cas9 system[J].Sci Rep,(5):10342.
SUN YG,ZHANG X,WU CY,et al.,2016.Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase[J].Mol Plant,9(4):628-631.
SVITASHEV S,YOUNG JK,SCHWARTZ C,et al.,2015.Targeted mutagenesis,precise gene editing,and site-specific gene insertion in maize using Cas9 and guide RNA[J].Plant Physiol,169(2):931-945.
UNCKLESS RL,CLARK AG,MESSER PW,2017.Evolution of resistance against CRISPR/Cas9 gene drive[J].Genetics,205(2):827.
VAN ZEIJL A,WARDHANI TAK,SEIFI KALHOR M,et al.,2018.CRISPR/Cas9-mediated mutagenesis of four putative symbiosis genes of the tropical tree Parasponia andersonii reveals novel phenotypes[J].Front Plant Sci,(9):284.
WANG LX,WANG LL,TAN Q,et al.,2016.Efficient inactivation of symbiotic nitrogen fixation related genes in lotus japonicas using CRISPR-Cas9[J].Front Plant Sci,7(703).
WANG MG,LU YM,BOTELLA JR,et al.,2017.Gene targeting by homology-directed repair in rice using ageminivirusbased CRISPR/Cas9 system[J].Mol Plant,10(7):1007-1010.
WANG S,ZHANG S,WANG W,et al.,2015.Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system[J].Plant Cell Rep,1473-1476(9):10342.
WEI Y,ZHANG XH,LI DL,2017.The“new favorite”of gene editing technology-single base editors[J].Hereditas,39(12):1115-1121.[魏瑜,张晓辉,李大力,2017.基因编辑之“新宠”-单碱基基因组编辑系统[J].遗传,39(12):1115-1121.]
XIE K,MINKENBERG B,YANG Y,2015.Boosting CRISPR/Cas9 multiplex editing capability with the endogenoust RNA-processing system[J].Proc Natl Acad Sci USA,112(11):3570-3575.
YANG H,WANG HY,SHIVALILA CS,et al.,2013.Onestep generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering[J].Cell,154(6):1370-1379.
YIN KQ,HAN T,LIU G,et al.,2015.Ageminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing[J].Sci Rep,7(5):14926.
ZHANG B,YANG X,YANG C,et al.,2016a.Exploiting the CRISPR/Cas9 system for targeted genome mutagenesis in Petunia[J].Sci Rep,(6):20315.
ZHANG D,LI Z,LI JF,2016b.Targeted gene manipulation in plants using the CRISPR/Cas technology[J].J Genet Genom,43(5):251-262.
ZHANG L,ZHOU Q,2014.CRISPR/Cas technology:A revolutionary approach for genome engineering[J].Sci Chin Life Sci,57(6):639-640.