Preprint Article Version 1 This version is not peer-reviewed

A Multiplex CRISPR/Cas9 System for Use as an Anti-BmNPV Therapeutic

Version 1 : Received: 5 November 2018 / Approved: 7 November 2018 / Online: 7 November 2018 (10:23:52 CET)

How to cite: Dong, Z.; Qin, Q.; Hu, Z.; Chen, P.; Huang, L.; Long, J.; Tian, T.; Lu, C.; Pan, M. A Multiplex CRISPR/Cas9 System for Use as an Anti-BmNPV Therapeutic. Preprints 2018, 2018110159 (doi: 10.20944/preprints201811.0159.v1). Dong, Z.; Qin, Q.; Hu, Z.; Chen, P.; Huang, L.; Long, J.; Tian, T.; Lu, C.; Pan, M. A Multiplex CRISPR/Cas9 System for Use as an Anti-BmNPV Therapeutic. Preprints 2018, 2018110159 (doi: 10.20944/preprints201811.0159.v1).

Abstract

Clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology guided by a single-guide RNA (sgRNA) has recently opened a new avenue for antiviral therapy. A unique capability of the CRISPR/Cas9 system is multiple genome engineering. However, there are few applications in insect viruses by a single Cas9 enzyme targeting two or more sgRNA at different genomic sites for simultaneous production of multiple DNA breaks. To address the need for multi-gene editing and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system to express multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells. Here, ie-1, gp64, lef-11, and dnapol genes were screened and identified as multiple sgRNA editing sites according to the BmNPV system infection and DNA replication mechanism. Furthermore, we constructed a multiplex editing vector sgMultiple to efficiently regulate multiplex gene editing steps and inhibit BmNPV replication after viral infection. This is the first report that describes the application of multiplex CRISPR/Cas9 system inhibiting insect virus replication. This multiplex system can significant enable the potential of CRISPR/Cas9-based multiplex genome engineering in transgenic silkworms.

Subject Areas

Bombyx mori; BmNPV; antiviral therapeutic; CRISPR/Cas9; multi-gene editing

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