清脆的
Cas9
基因组编辑
生物
亚基因组mRNA
多路复用
基因
引导RNA
计算生物学
CRISPR干扰
基因组
遗传学
作者
Zhanqi Dong,Qin Qi,Zhigang Hu,Peng Chen,Liang Huang,Xinling Zhang,Ting Tian,Cheng Lü,Min‐Hui Pan
标识
DOI:10.1007/s12250-019-00121-4
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.
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