CRISPR/Cas9‐mediated 2‐sgRNA cleavage facilitates Pseudorabies virus editing

Abstract

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. FASEB J . 32, 4293–4301 (2018). www.fasebj.org