文章基本信息

标题：A surrogate reporter system for multiplexable evaluation of CRISPR/Cas9 in targeted mutagenesis
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作者：Hongmin Zhang ; Yuexin Zhou ; Yinan Wang 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2018
卷号：8
期号：1
页码：1042
DOI：10.1038/s41598-018-19317-x
语种：English
出版社：Springer Nature
摘要：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.