茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建
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摘要
CRISPR/Cas9技术是一门新兴的基因组定点编辑技术,具有操作简单、高效的优点,可轻松实现对目标基因的敲除、替换和定点突变等操作。该技术刚诞生,就受到了全球生命科学领域研究者的关注,不到3年的时间就已经成功应用于多种动、植物当中。然而CRISPR/Cas9技术在茶树中的应用面临载体构建问题,本文以茶树咖啡碱合成酶为例,联合采用常规PCR、Overlapping PCR和Golden Gate Cloning技术,构建了包含茶树咖啡碱合成酶双靶点的CRISPR/Cas9基因编辑载体,为CRISPR/Cas9介导的基因组编辑技术在茶树中的应用奠定了坚实基础。
CRISPR/Cas9 technology(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) is a novel and powerful approach for targeted genome editing, such as targeted gene knock out or site-directed mutagenesis in a simple and easy way. Since its establishment, the CRISPR/Cas9 technique has been successfully applied in many eukaryotic organisms, including more than 10 plant species. However, it has not been available for genome editing of tea plant [Camellia sinensis(L.) O. Kuntze] due to the difficulty in constructing CRISPR/Cas9 expression vector. The present work developed an efficient method to construct a CRISPR/Cas9 expression vector for genome editing a tea caffeine synthase(TCS) by using general PCR, overlapping PCR and golden gate cloning technology. The present work would promote the application of CRISPR/Cas9 technology in genomic modification in tea plants.
CRISPR/Cas9 technology(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) is a novel and powerful approach for targeted genome editing, such as targeted gene knock out or site-directed mutagenesis in a simple and easy way. Since its establishment, the CRISPR/Cas9 technique has been successfully applied in many eukaryotic organisms, including more than 10 plant species. However, it has not been available for genome editing of tea plant [Camellia sinensis(L.) O. Kuntze] due to the difficulty in constructing CRISPR/Cas9 expression vector. The present work developed an efficient method to construct a CRISPR/Cas9 expression vector for genome editing a tea caffeine synthase(TCS) by using general PCR, overlapping PCR and golden gate cloning technology. The present work would promote the application of CRISPR/Cas9 technology in genomic modification in tea plants.
引文
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[2]Liu SC,Jin JQ,Ma JQ,et al.Transcriptomic analysis of tea plant responding to drought stress and recovery[J].PLo S ONE,2016,11(1):e0147306.http://dx.doi.org/10.1371/journal.pone.0147306.
[3]Wei Y,Jing W,Youxiang Z,et al.Genome-wide identification of genes probably relevant to the uniqueness of tea plant(Camellia sinensis)and its cultivars[J].International Journal of Genomics,2015,2015:527054.doi:10.1155/2015/527054.
[4]Wang XC,Zhao QY,Ma CL,et al.Global transcriptome profiles of Camellia sinensis during cold acclimation[J].BMC Genomics 2013,14:415.DOI:10.1186/1471-2164-14-415.
[5]Mukhopadhyay M,Mondal TK,Chand PK.Biotechnological advances in tea(Camellia sinensis[L.]O.Kuntze):a review[J].Plant Cell Reports,2015,35(2):255-287.
[6]Fauser F,Roth N,Pacher M,et al.In planta gene targeting[J].Proc Natl Acad Sci USA,2012,109(19):7535-7540.
[7]Li T,Liu B,Spalding MH,et al.High-efficiency TALEN-based gene editing produces disease-resistant rice[J].Nat Biotech,2012,30(5):390-392.
[8]Belhaj K,Chaparro-Garcia A,Kamoun S,et al.Editing plant genomes with CRISPR/Cas9[J].Current Opinion in Biotechnology,2015,32:76-84.
[9]Mali P,Yang LH,Esvelt KM,et al.RNA-Guided human genome engineering via Cas9[J].Science,2013,339(6121):823-826.
[10]Xie K,Yang Y.RNA-guided genome editing in plants using a CRISPR-Cas system[J].Mol Plant,2013,6(6):1975-1983.
[11]Schaeffer SM,Nakata PA.CRISPR/Cas9-mediated genome editing and gene replacement in plants:transitioning from lab to field[J].Plant Sci,2015,240:130-142.
[12]Wiedenheft B,Sternberg SH,Doudna JA.RNA-guided genetic silencing systems in bacteria and archaea[J].Nature,2012,482(7385):331-338.
[13]Jinek M,Chylinski K,Fonfara I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,2012,337(6096):816-821.
[14]Wang HY,Yang H,Shivalila CS,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.
[15]Schiml S,Puchta H.Revolutionizing plant biology:multiple ways of genome engineering by CRISPR/Cas[J].Plant Methods,2016,12(1):1-9.
[16]Lintner NG,Frankel KA,Tsutakawa SE,et al.The structure of the crispr-associated protein csa3 provides insight into the regulation of the CRISPR/Cas system[J].Journal of Molecular Biology,2011,405(4):939-955.
[17]Gilbert LA,Larson MH,Morsut L,et al.CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[J].Cell,2013,154(2):442-451.
[18]Nekrasov V,Staskawicz B,Weigel D,et al.Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease[J].Nat Biotech,2013,31(8):691-693.
[19]Jiang WZ,Zhou HB,Bi HH,et al.Demonstration of CRISPR/Cas9/sg RNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice[J].Nucleic Acids Res,2013,41(20):e188.
[20]Wang Z,Xing H,Dong L,et al.Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation[J].Genome Biol,2015,16:144.
[21]Upadhyay SJ,Alok A,Tuli R.RNA-guided genome editing for target gene mutations in wheat[J].G3(Bethesda).2013,3(12):2233-2238.DOI:10.1534/g3.113.008847.
[22]Liang Z,Zhang K,Chen KL,et al.Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system[J].J Genetics Genomics,2014,41(2):63-68.
[23]Zhang H,Zhang J,Wei P,et al.The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation[J].Plant Biotechnol J,2014,12(6):797-807.
[24]?ermák T,Baltes NJ,?egan R,et al.High-frequency,precise modification of the tomato genome[J].Genome Biol,2015,16:232.DOI:10.1186/s13059-015-0796-9.
[25]Jia H,Wang N.Targeted genome editing of sweet orange using Cas9/sg RNA[J].PLo S ONE,2014,9(4):e93806.DOI:10.1371/journal.pone.0093806.
[26]Fan D,Liu T,Li C,et al.Efficient CRISPR/Cas9-mediated targeted mutagenesis in populus in the first generation[J].Scientific Reports,2015,5:12217.
[27]Zhang B,Yang X,Yang C,et al.Exploiting the CRISPR/Cas9 system for targeted genome mutagenesis in petunia[J].Scientific Reports,2016,6:20315.
[28]Ma X,Zhang Q,Zhu Q,et al.A Robust CRISPR/Cas9system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants[J].Mol Plant,2015,8(8):1274-1284.
[29]Marillonnet S,Werner S.Assembly of multigene constructs using golden gate cloning[J].Methods Mol Biol,2015,1321:269-284.