CRISPR质粒和同源重组模板的共转染可降低CRISPR/Cas9系统的基因编辑效率

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英文篇名：Co-delivery of CRISPR plasmid and HDR donor can reduce the gene editing efficiency of CRISPR/Cas9 system 作者：林琼 ; 张祝琴 ; 刘德培 英文作者：LIN Qiong;ZHANG Zhu-qin;LIU De-pei;State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC; 关键词：CRISPR/Cas9系统 ; 共转染 ; 分开转染 ; 同源重组模板 英文关键词：CRISPR/Cas9 system;;co-transfection;;sequential transfection;;homology-directed repair template 中文刊名：基础医学与临床 英文刊名：Basic & Clinical Medicine 机构：中国医学科学院基础医学研究所北京协和医学院基础学院医学分子生物学国家重点实验室; 出版日期：2019-07-05 出版单位：基础医学与临床 年：2019 期：07 基金：中国医学科学院医学与健康科技创新工程(2017-I2M-B&R-04);中国医学科学院医学表观遗传中心基金(2017PT31035) 语种：中文; 页：130-136 页数：7 CN：11-2652/R ISSN：1001-6325 分类号：Q78

摘要

目的探讨CRISPR质粒和同源重组模板的共转染对CRISPR/Cas9系统的基因编辑效率的影响以及可行的解决办法。方法用T7E1内切酶实验和RT-qPCR检测只转染CRISPR质粒组和质粒和模板共转染组细胞内Cas9的切割效率,Cas9 RNA和DNA水平的差异;RT-qPCR检测降低共转染的模板DNA的数量时,细胞内Cas9 DNA和RNA水平的变化;RT-qPCR和T7E1内切酶实验检测先转染CRISPR质粒后转染同源重组模板时,细胞内Cas9 DNA和RNA水平以及切割效率的变化。结果与只转染CRISPR质粒组相比,CRISPR质粒和同源重组模板的共转染显著降低了CRISPR质粒的转染效率(P<0.001)和Cas9的切割效率(P<0.001)。而先转染质粒后转染同源重组模板组和只转染质粒组在CRISPR质粒的转染效率和Cas9的切割效率差别无统计学意义。结论 CRISPR质粒和同源重组模板的共转染降低了CRISPR/Cas9系统的基因编辑效率,而先转染质粒后转染同源重组模板可以解决这一问题。
        Objective To explore the effect of co-transfection of CRISPR plasmid and HDR donors on the gene editing efficiency of CRISPR/Cas9 system and a feasible solution to the problem. Methods T7 E1 assay and RT-qPCR were used to compare the cleavage efficiency of Cas9 protein, Cas9 RNA and DNA level between cells transfected with CRSIPR plasmid only and cells co-transfected with CRISPR plasmid and HDR templates; RT-qPCR was used to detect the changes of intracellular Cas9 DNA and RNA level when reducing the quantity of co-transfected template DNA; RT-qPCR and T7 E1 assay were used to measure the DNA level, RNA level and cleavage activity of Cas9 when delivering CRISPR plasmid followed by HDR donors. Results Compared with the group only CRISPR plasmid transfected, co-transfection of CRISPR plasmid and HDR donors significantly reduced the transfectionefficiency of CRISPR plasmid(P<0.001) and cleavage activity of Cas9(P<0.001). But there was no significant difference in transfection efficiency of CRISPR plasmid and cutting efficiency of CRISPR/Cas9 system among only CRISPR plasmid transfected group and groups sequentially transfected with CRISPR plasmid and HDR donors. Conclusions Co-transfection of CRISPR plasmid and HDR donors can reduce the gene editing efficiency of CRISPR/Cas9 system and this problem can be solved by delivering CRISPR plasmid first and then HDR templates.

引文

[1] Ran FA,Hsu PD,Wright J,et al.Genome engineering using the CRISPR-Cas9 system[J].Nature protocols,2013,8:2281-2308.
    [2] Salsman J,Dellaire G.Precision genome editing in the CRISPR era[J].Biochem and Cell Biol,2017,95:187-201.
    [3] Shrivastav M,De Haro LP,Nickoloff JA.Regulation of DNA double-strand break repair pathway choice[J].Cell Res,2008,18:134-147.
    [4] Liu M,Rehman S,Tang X,et al.Methodologies for improving HDR efficiency[J].Frontiers in genetics,2018,9:691.doi:10.3389/fgene.2018.00691.
    [5] Chu VT,Weber T,Wefers B,et al.Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells[J].Nature Biotechnol,2015,33:543-548.
    [6] Maruyama T,Dougan SK,Truttmann MC,et al.Increas-ing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining[J].Nature Biotechnol,2015,33:538-542.
    [7] Richardson CD,Ray GJ,DeWitt MA,et al.Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA[J].Nature Biotechnol,2016,34:339-344.
    [8] Li G,Liu D,Zhang X,et al.Suppressing Ku70/Ku80 expression elevates homology-directed repair efficiency in primary fibroblasts[J].Int J Biochem Cell B,2018,99:154-160.
    [9] Charpentier M,Khedher AHY,Menoret S,et al.CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair[J].Nature Commun,2018,9:1133.doi:10.1038/s41467-018-03475-7.
    [10] Liang X,Potter J,Kumar S,et al.Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA,Cas9 nuclease,and donor DNA[J].J Biotechnol,2017,241:136-146.
    [11] Mali P,Yang L,Esvelt KM,et al.RNA-guided human genome engineering via Cas9[J].Science,2013,339:823-826.
    [12] Oceguera-Yanez F,Kim SI,Matsumoto T,et al.Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives[J].Methods,2016,101:43-55.