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CRISPR/Cas9 Cleavage of Viral DNA Efficiently Suppresses Vaccinia Viruses
Wang Jiaojiao1, Zhang Xinmin1, Ni Aiming2, Zhang Kangjian2, Liu Xinyuan1,2, Liu Xijun3*
1Xin Yuan Institute of Medicine and Biotechnology, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China; 2Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; 3Institute of Pan-vascular Diseases, Shanghai Tenth People′s Hospital, Tongji University, Shanghai 200072, China
Abstract: Previous study had shown that viral genomes could be edited effectively using CRISPR/Cas9-mediated genomic editing technique. However， the precise edit to viruses with large DNA genomes， such as vaccinia viruses by CRISPR/Cas9 system has not been reported. To explore the effects of genetic inactivation or mutation on vaccinia viral replication， the recombinant virus armed with an nonessential enhanced green fluorescent protein (EGFP) gene (WR-EGFP) was selected as the object of study here. Two guide RNAs were designed to target the coding region of EGFP gene. Fluorescence microscopy result showed that expression of EGFP significantly decreased in cells that both infected with WR-EGFP viruses and transfected with Cas9/gRNA plasmids， compared with control group that only infected with WR-EGFP. The Cas9/gRNA could induce site-specific cleavage of EGFP gene， thus caused mismatch repair of DNA viral genomes which were detected by SURVEYOR assay. Moreover，the mismatch repair of DNA sequence in virus resulted in losing the ability of virus replication. Taken together， the CRISPR/Cas9 system was a highly efficient strategy for editing the recombinant vaccinia viral genomes which could significantly inhibit the replication of virus.
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