Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing.

Abstract:

:The clustered regularly interspaced short palindromic repeats (CRISPR) system-associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9-single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock-ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock-ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in baculovirus, contributing to the engineering of baculovirus as a protein expression vector as well as a biological control agent.

journal_name

Biotechnol Bioeng

authors

Pazmiño-Ibarra V,Mengual-Martí A,Targovnik AM,Herrero S

doi

10.1002/bit.27139

subject

Has Abstract

pub_date

2019-11-01 00:00:00

pages

2823-2833

issue

11

eissn

0006-3592

issn

1097-0290

journal_volume

116

pub_type

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