A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 'self-cleaving' GFP-expression plasmid.
A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 'self-cleaving' GFP-expression plasmid.
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DOI：
10.1093/dnares/dsx029
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发表时间：
2017-12-01
期刊：
影响因子：
--
通讯作者：
Welker E
中科院分区：
文献类型：
--
作者：
Tálas A;Kulcsár PI;Weinhardt N;Borsy A;Tóth E;Szebényi K;Krausz SL;Huszár K;Vida I;Sturm Á;Gordos B;Hoffmann OI;Bencsura P;Nyeste A;Ligeti Z;Fodor E;Welker E
引导RNA（gRNA）（结合并决定化脓链球菌Cas9核酸酶的序列特异性的短RNA分子）介导DNA切割的功效显著变化。因此，选择合适的靶位点以及因此选择间隔区序列对于大多数应用是至关重要的。在这里，我们描述了一种简单的，无与伦比的方法，用于实验性地预先测试靶向基因的各种gRNA的效率。该方法探索了NHEJ克隆、无同源臂和细胞内线性化的GFP表达质粒的基因组整合。当应用该方法时，使用含有通用gRNA和相应靶标的“自切割”GFP质粒减轻了克隆负担。这些通用gRNA介导有效的质粒切割，并被设计为避免几种模型物种中的基因组靶标。该方法结合了通过应用荧光报告系统提供的直接FACS检测和能够在其基因组背景下测试靶的基于PCR的方法的优点，而不需要任何额外的克隆步骤。此外，我们表明，NHEJ克隆也可以用于哺乳动物细胞中的供体质粒的靶向整合高达10 kb的大小，高达30%的效率，没有任何选择或富集。
The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell. The use of ‘self-cleaving’ GFP-plasmids containing universal gRNAs and corresponding targets alleviates cloning burdens when this method is applied. These universal gRNAs mediate efficient plasmid cleavage and are designed to avoid genomic targets in several model species. The method combines the advantages of the straightforward FACS detection provided by applying fluorescent reporter systems and of the PCR-based approaches being capable of testing targets in their genomic context, without necessitating any extra cloning steps. Additionally, we show that NHEJ-cloning can also be used in mammalian cells for targeted integration of donor plasmids up to 10 kb in size, with up to 30% efficiency, without any selection or enrichment.
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影响因子：
7
作者：
Auer TO;Duroure K;De Cian A;Concordet JP;Del Bene F
通讯作者：
Del Bene F
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
46.9
作者：
Hockemeyer, Dirk;Soldner, Frank;Beard, Caroline;Gao, Qing;Mitalipova, Maisam;DeKelver, Russell C.;Katibah, George E.;Amora, Ranier;Boydston, Elizabeth A.;Zeitler, Bryan;Meng, Xiangdong;Miller, Jeffrey C.;Zhang, Lei;Rebar, Edward J.;Gregory, Philip D.;Urnov, Fyodor D.;Jaenisch, Rudolf
通讯作者：
Jaenisch, Rudolf
影响因子：
3.8
作者：
Cristea, Sandra;Freyvert, Yevgeniy;Cost, Gregory J.
通讯作者：
Cost, Gregory J.
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo