A surrogate reporter system for multiplexable evaluation of CRISPR/Cas9 in targeted mutagenesis.
A surrogate reporter system for multiplexable evaluation of CRISPR/Cas9 in targeted mutagenesis.
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用于在靶向诱变中对 CRISPR/Cas9 进行多重评估的替代报告系统。
DOI：
10.1038/s41598-018-19317-x
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发表时间：
2018-01-18
影响因子：
4.6
通讯作者：
Wei W
中科院分区：
文献类型：
--
作者：
Zhang H;Zhou Y;Wang Y;Zhao Y;Qiu Y;Zhang X;Yue D;Zhou Z;Wei W
基因组编辑中的工程化核酸酶在不同的靶基因座表现出不同的效率。因此，不断需要评估给定基因座的突变率。T7核酸内切酶1（T7 E1）和Surveyor错配切割测定是最广泛使用的方法，但是它们是劳动和耗时的，特别是当必须平行处理多个样品时。在这里，我们报告了一个替代系统，称为UDAR（通用供体报告），以评估CRISPR/Cas9在靶向诱变中的效率。基于非同源末端连接（NHEJ）介导的敲入策略，基于UDAR的测定允许我们快速评估sgRNA的靶向效率。通过一步转染和荧光激活细胞分选（FACS）分析，UDAR测定可以在三天内大规模完成。为了检测CRISPR/Cas9系统产生的突变，在UDAR和T7 E1测定的结果之间观察到显著的正相关性。一致地，UDAR测定可以定量评估博来霉素或ICRF 193诱导的双链断裂（DSB），这表明这种新策略广泛适用于评估各种试剂的DSB诱导能力。随着基因组编辑在生物医学研究中的影响越来越大，UDAR方法可以显着有利于靶向诱变的评估，特别是用于高通量目的。
Engineered nucleases in genome editing manifest diverse efficiencies at different targeted loci. There is therefore a constant need to evaluate the mutation rates at given loci. T7 endonuclease 1 (T7E1) and Surveyor mismatch cleavage assays are the most widely used methods, but they are labour and time consuming, especially when one must address multiple samples in parallel. Here, we report a surrogate system, called UDAR (Universal Donor As Reporter), to evaluate the efficiency of CRISPR/Cas9 in targeted mutagenesis. Based on the non-homologous end-joining (NHEJ)-mediated knock-in strategy, the UDAR-based assay allows us to rapidly evaluate the targeting efficiencies of sgRNAs. With one-step transfection and fluorescence-activated cell sorting (FACS) analysis, the UDAR assay can be completed on a large scale within three days. For detecting mutations generated by the CRISPR/Cas9 system, a significant positive correlation was observed between the results from the UDAR and T7E1 assays. Consistently, the UDAR assay could quantitatively assess bleomycin- or ICRF193-induced double-strand breaks (DSBs), which suggests that this novel strategy is broadly applicable to assessing the DSB-inducing capability of various agents. With the increasing impact of genome editing in biomedical studies, the UDAR method can significantly benefit the evaluation of targeted mutagenesis, especially for high-throughput purposes.
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影响因子：
5.8
作者：
Park, Jeongbin;Bae, Sangsu;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
12.3
作者：
Dang Y;Jia G;Choi J;Ma H;Anaya E;Ye C;Shankar P;Wu H
通讯作者：
Wu H
一种新型的自动定量方法，用于对DNA双链破裂响应进行高素质筛选分析。
影响因子：
4.6
作者：
Feng J;Lin J;Zhang P;Yang S;Sa Y;Feng Y
通讯作者：
Feng Y
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：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle