利用dCas9-FokⅠ编辑大鼠Dnmt1基因
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摘要
CRISPR/Cas9的发现为多种生物的基因编辑提供了强有力的工具。然而,该系统在提供靶向性基因修饰的同时,会产生一些不需要的突变,即脱靶现象。为提高CRISPR/Cas9的特异性,我们将野生型Fok I核酸内切酶的功能结构域与催化功能区失活的Cas9蛋白(dCas9)进行融合,形成融合蛋白用于降低脱靶效应。FokⅠ是一种依赖于二聚化才能行使内切酶活性的核酸酶,在本研究中,通过将FokⅠ功能结构融合到dCas9的N端,构建表达质粒p ST1374-dCas9-FokⅠ。我们前期研究中,发现一个sgRNA在介导Cas9编辑Dnmt1基因建立条件敲除大鼠时,存在显著的脱靶现象。以此为基础,我们利用dCas9-FokⅠ/sgRNA系统编辑大鼠Dnmt1基因,研究该系统是否能够进行基因编辑以及是否能够提高基因编辑特异性。将转录好的dCas9-FokⅠmRNA和sgRNA显微注射到SD大鼠的受精卵中,用于产生基因编辑大鼠。通过显微注射以及胚胎移植,最终获得43只F0代大鼠,其中两只在靶点位置包含突变,突变效率达4.5%。对脱靶情况进行分析,结果显示,无脱靶现象存在。综上,表明dCas9-FokⅠ/sgRNA可以应用于编辑大鼠基因,并能显著提高特异性。尽管dCas9-FokⅠ/sgRNA系统相比于Cas9/sgRNA系统,基因编辑效率有所下降,但是该技术的发展为基因治疗提供了可供选择的潜在工具。
CRISPR/Cas9 system provides a powerful tool for genome editing in various organisms.However,this system also generates some unwanted mutations while modifying the defined target sites.In order to improve the specificity of CIRSPR/Cas9 system,we used the catalytically inactive Cas9(dCas9)fused with a wild type FokⅠ nuclease domain to reduce the off-target effects.FokⅠ is a dimerization dependent restriction endonuclease.In this study,we constructed a dCas9-FokⅠ expression plasmid named p ST1374-dCas9-FokⅠ by fusing the wild type FokⅠ nuclease domain to the N terminus of dCas9.In previous work,we obtained a sgRNA targeting site with high off-target effects when producing Dnmt1 conditional knockout rat.In this study,the sgRNA target site of Dnmt1 was used in dCas9-FokⅠ/sgRNAsystem to generate Dnmt1 gene knockout rats.The transcribed dCas9-FokⅠ mRNA and sgRNA were mixed and microinjected into one-cell embryos of SD rats to generate rats with target mutations.Fortythree F0 rats were obtained after microinjection and embryo transfer.Two rats with indel modifications at the target locus were obtained with the efficiency of 4.5%.No real unwanted mutation was detected according to off-target analysis.Our results demonstrate that the dCas9-FokⅠ/sgRNA can be used as a genome editing tool for rat with improved target specificity.Although genome editing with dCas9-FokⅠ is with lower efficiency compared with wild-type Cas9/sgRNA system,our data suggest that the dCas9-FokⅠ/sgRNA system provides a potential alternative tool for gene therapy.
CRISPR/Cas9 system provides a powerful tool for genome editing in various organisms.However,this system also generates some unwanted mutations while modifying the defined target sites.In order to improve the specificity of CIRSPR/Cas9 system,we used the catalytically inactive Cas9(dCas9)fused with a wild type FokⅠ nuclease domain to reduce the off-target effects.FokⅠ is a dimerization dependent restriction endonuclease.In this study,we constructed a dCas9-FokⅠ expression plasmid named p ST1374-dCas9-FokⅠ by fusing the wild type FokⅠ nuclease domain to the N terminus of dCas9.In previous work,we obtained a sgRNA targeting site with high off-target effects when producing Dnmt1 conditional knockout rat.In this study,the sgRNA target site of Dnmt1 was used in dCas9-FokⅠ/sgRNAsystem to generate Dnmt1 gene knockout rats.The transcribed dCas9-FokⅠ mRNA and sgRNA were mixed and microinjected into one-cell embryos of SD rats to generate rats with target mutations.Fortythree F0 rats were obtained after microinjection and embryo transfer.Two rats with indel modifications at the target locus were obtained with the efficiency of 4.5%.No real unwanted mutation was detected according to off-target analysis.Our results demonstrate that the dCas9-FokⅠ/sgRNA can be used as a genome editing tool for rat with improved target specificity.Although genome editing with dCas9-FokⅠ is with lower efficiency compared with wild-type Cas9/sgRNA system,our data suggest that the dCas9-FokⅠ/sgRNA system provides a potential alternative tool for gene therapy.
引文
[1]Hwang WY,Fu Y,Reyon D,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system[J].Nat Biotechnol,2013,31(3):227-229
[2]Ma Y,Zhang X,Shen B,et al.Generating rats with conditional alleles using CRISPR/Cas9[J].Cell Res,2014,24(1):122-125
[3]Shen B,Zhang J,Wu H,et al.Generation of gene-modified mice via Cas9/RNA-mediated gene targeting[J].Cell Res,2013,23(5):720-723
[4]Niu Y,Shen B,Cui Y,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
[5]Wang H,Yang H,Shivalila CS,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Casmediated genome engineering[J].Cell,2013,153(4):910-918
[6]Li D,Qiu Z,Shao Y,et al.Heritable gene targeting in the mouse and rat using a CRISPR-Cas system[J].Nat Biotechnol,2013,31(8):681-683
[7]Bedell VM,Wang Y,Campbell JM,et al.In vivo genome editing using a high-efficiency TALEN system[J].Nature,2012,491(7422):114-118
[8]Tesson L,Usal C,Menoret S,et al.Knockout rats generated by embryo microinjection of TALENs[J].Nat Biotechnol,2011,29(8):695-696
[9]Doyon Y,Mc Cammon JM,Miller JC,et al.Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases[J].Nat Biotechnol,2008,26(6):702-708
[10]Meng X,Noyes MB,Zhu LJ,et al.Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases[J].Nat Biotechnol,2008,26(6):695-701
[11]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
[12]Yin H,Xue W,Chen S,et al.Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype[J].Nat Biotechnol,2014,32(6):551-553
[13]Fu Y,Foden JA,Khayter C,et al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells[J].Nat Biotechnol,2013,31(9):822-826
[14]Fu Y,Sander JD,Reyon D,et al.Improving CRISPR-Cas nuclease specificity using truncated guide RNAs[J].Nat Biotechnol,2014,32(3):279-284
[15]Ran FA,Hsu PD,Lin CY,et al.Double nicking by RNAguided CRISPR Cas9 for enhanced genome editing specificity[J].Cell,2013,154(6):1380-1389
[16]Shen B,Zhang W,Zhang J,et al.Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects[J].Nat Methods,2014,11(4):399-402
[17]Mali P,Aach J,Stranges PB,et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering[J].Nat Biotechnol,2013,31(9):833-838
[18]Tsai SQ,Wyvekens N,Khayter C,et al.Dimeric CRISPR RNAguided Fok I nucleases for highly specific genome editing[J].Nat Biotechnol,2014,32(6):569-576
[19]Guilinger JP,Thompson DB,Liu DR.Fusion of catalytically inactive Cas9 to Fok I nuclease improves the specificity of genome modification[J].Nat Biotechnol,2014,32(6):577-582
[20]Ma Y,Ma J,Zhang X,et al.Generation of e GFP and Cre knockin rats by CRISPR/Cas9[J].FEBS J,2014,281(17):3779-3790
[21]Ma Y,Shen B,Zhang X,et al.Heritable multiplex genetic engineering in rats using CRISPR/Cas9[J].PLo S One,2014,9(3):e89413
[22]Abbott A.Laboratory animals:the Renaissance rat[J].Nature,2004,428(6982):464-466
[23]Tong C,Li P,Wu NL,et al.Production of p53 gene knockout rats by homologous recombination in embryonic stem cells[J].Nature,2010,467(7312):211-213
[24]Geurts AM,Cost GJ,Freyvert Y,et al.Knockout rats via embryo microinjection of zinc-finger nucleases[J].Science,2009,325(5939):433
[25]Li W,Teng F,Li T,et al.Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems[J].Nat Biotechnol,2013,31(8):684-686
[26]Hsu PD,Scott DA,Weinstein JA,et al.DNA targeting specificity of RNA-guided Cas9 nucleases[J].Nat Biotechnol,2013,31(9):827-832
[27]Cho SW,Kim S,Kim Y,et al.Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases[J].Genome Res,2014,24(1):132-141
[28]Slaymaker IM,Gao L,Zetsche B,et al.Rationally engineered Cas9 nucleases with improved specificity[J].Science,2016,351(6268):84-88
[29]Kleinstiver BP,Pattanayak V,Prew MS,et al.High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide offtarget effects[J].Nature,2016,529(7587):490-495
[2]Ma Y,Zhang X,Shen B,et al.Generating rats with conditional alleles using CRISPR/Cas9[J].Cell Res,2014,24(1):122-125
[3]Shen B,Zhang J,Wu H,et al.Generation of gene-modified mice via Cas9/RNA-mediated gene targeting[J].Cell Res,2013,23(5):720-723
[4]Niu Y,Shen B,Cui Y,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
[5]Wang H,Yang H,Shivalila CS,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Casmediated genome engineering[J].Cell,2013,153(4):910-918
[6]Li D,Qiu Z,Shao Y,et al.Heritable gene targeting in the mouse and rat using a CRISPR-Cas system[J].Nat Biotechnol,2013,31(8):681-683
[7]Bedell VM,Wang Y,Campbell JM,et al.In vivo genome editing using a high-efficiency TALEN system[J].Nature,2012,491(7422):114-118
[8]Tesson L,Usal C,Menoret S,et al.Knockout rats generated by embryo microinjection of TALENs[J].Nat Biotechnol,2011,29(8):695-696
[9]Doyon Y,Mc Cammon JM,Miller JC,et al.Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases[J].Nat Biotechnol,2008,26(6):702-708
[10]Meng X,Noyes MB,Zhu LJ,et al.Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases[J].Nat Biotechnol,2008,26(6):695-701
[11]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
[12]Yin H,Xue W,Chen S,et al.Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype[J].Nat Biotechnol,2014,32(6):551-553
[13]Fu Y,Foden JA,Khayter C,et al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells[J].Nat Biotechnol,2013,31(9):822-826
[14]Fu Y,Sander JD,Reyon D,et al.Improving CRISPR-Cas nuclease specificity using truncated guide RNAs[J].Nat Biotechnol,2014,32(3):279-284
[15]Ran FA,Hsu PD,Lin CY,et al.Double nicking by RNAguided CRISPR Cas9 for enhanced genome editing specificity[J].Cell,2013,154(6):1380-1389
[16]Shen B,Zhang W,Zhang J,et al.Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects[J].Nat Methods,2014,11(4):399-402
[17]Mali P,Aach J,Stranges PB,et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering[J].Nat Biotechnol,2013,31(9):833-838
[18]Tsai SQ,Wyvekens N,Khayter C,et al.Dimeric CRISPR RNAguided Fok I nucleases for highly specific genome editing[J].Nat Biotechnol,2014,32(6):569-576
[19]Guilinger JP,Thompson DB,Liu DR.Fusion of catalytically inactive Cas9 to Fok I nuclease improves the specificity of genome modification[J].Nat Biotechnol,2014,32(6):577-582
[20]Ma Y,Ma J,Zhang X,et al.Generation of e GFP and Cre knockin rats by CRISPR/Cas9[J].FEBS J,2014,281(17):3779-3790
[21]Ma Y,Shen B,Zhang X,et al.Heritable multiplex genetic engineering in rats using CRISPR/Cas9[J].PLo S One,2014,9(3):e89413
[22]Abbott A.Laboratory animals:the Renaissance rat[J].Nature,2004,428(6982):464-466
[23]Tong C,Li P,Wu NL,et al.Production of p53 gene knockout rats by homologous recombination in embryonic stem cells[J].Nature,2010,467(7312):211-213
[24]Geurts AM,Cost GJ,Freyvert Y,et al.Knockout rats via embryo microinjection of zinc-finger nucleases[J].Science,2009,325(5939):433
[25]Li W,Teng F,Li T,et al.Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems[J].Nat Biotechnol,2013,31(8):684-686
[26]Hsu PD,Scott DA,Weinstein JA,et al.DNA targeting specificity of RNA-guided Cas9 nucleases[J].Nat Biotechnol,2013,31(9):827-832
[27]Cho SW,Kim S,Kim Y,et al.Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases[J].Genome Res,2014,24(1):132-141
[28]Slaymaker IM,Gao L,Zetsche B,et al.Rationally engineered Cas9 nucleases with improved specificity[J].Science,2016,351(6268):84-88
[29]Kleinstiver BP,Pattanayak V,Prew MS,et al.High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide offtarget effects[J].Nature,2016,529(7587):490-495