CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing

文献类型： 外文期刊

第一作者： Tang, Yan-Dong

作者： Tang, Yan-Dong;Guo, Jin-Chao;Wang, Tong-Yun;Zhao, Kuan;Liu, Ji-Ting;Gao, Jia-Cong;Tian, Zhi-Jun;An, Tong-Qing;Cai, Xue-Hui

作者机构：

关键词： herpesvirus; knockout; knockin; recombination; DNA virus

期刊名称：FASEB JOURNAL （ 影响因子：5.191； 五年影响因子：5.955 ）

ISSN： 0892-6638

年卷期： 2018 年 32 卷 8 期

页码：

收录情况： SCI

摘要： Several groups have used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for DNA virus editing. In most cases, one single-guide RNA (sgRNA) is used, which produces inconsistencies in gene editing. In this study, we used a swine herpesvirus, pseudorabies virus, as a model to systematically explore the application of CRISPR/Cas9 in DNA virus editing. In our current report, we demonstrated that cotransfection of 2 sgRNAs and a viral genome resulted in significantly better knockout efficiency than the transfection-infection-based approach. This method could result in 100% knockout of 3500 bp of viral nonessential large fragments. Furthermore, knockin efficiency was significantly improved by using 2 sgRNAs and was also correlated with the number of background viruses. We also demonstrated that the background viruses were all 2-sgRNA-mediated knockout mutants. Finally, this study demonstrated that the efficacy of gene knockin is determined by the replicative kinetics of background viruses. We propose that CRISPR/Cas9 coupled with 2 sgRNAs creates a powerful tool for DNA virus editing and offers great potential for future applications.Tang, Y.-D., Guo, J.-C., Wang, T.-Y., Zhao, K., Liu, J.-T., Gao, J.-C., Tian, Z.-J., An, T.-Q., Cai, X.-H. CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

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