Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in Silkworms

• 来源期刊: 中国病毒学
• 年，卷（期）: 2019,（4）
• 页码范围: 444-453
• 页数: 10页
• 语种: 英文

摘要

Recently the developed single guide (sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology has opened a new avenue for antiviral therapy.The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously.However,there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses.To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses,we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells.We screened the immediate-early-1 gene (ie-1),the major envelope glycoprotein gene (gp64),and the late expression factor gene (lef-11),and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis.In addition,we constructed a multiplex editing vector (PSL1180-Cas9-sgIE1-sgLEF11-sgGP64,sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection.This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication.This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.