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

Abstract

adaptation of this methodology for the generation of viral knocks-in specific regions of the viral 35 genome. Analysis of the generated mutants revealed that the edition efficiency and the type of 36 changes was variable but relatively high. Depending on the targeted gene, the rate of edition 37 ranged from 10% to 40%. This study established the first report revealing the potential of 38 CRISPR/Cas9 for the edition of baculovirus contributing to the engineering of baculovirus as 39 protein expression vector as well as a biological control agent. 40 Keywords: CRISPR/Cas9, baculovirus, AcMNPV, genome editing, knock-out, knock-in 41 42 43 with the virus (3). During their viral biphasic life cycle, baculoviruses produce two distinct virion 49 phenotypes: occlusion derived viruses (ODV) and budded viruses (BV). While ODV are 50 involved in horizontal virus transmission from insect to insect through structures named 51 occlusion bodies (OB) which have the virus embedded within, BV are involved in spread of the 52 infection from cell to cell (4). 53Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the prototypic baculovirus 54 most common used for biotechnological purpose, has a circular genome of about 134 Kb that 55contains 156 predicted open reading frames (ORF) (5). The baculovirus genomes, and specially 56 AcMNPV genome, have been genetically modified in order to enhance their pesticide potency 57 and increase the quality and quantity of the recombinant protein expressed in the system (6, 7). 58These modifications include either the deletion of non-essential genes for virus survival or 59 infectivity and the insertion of foreign genes. For instance, in term of increase the insecticidal 60 activity of baculovirus, the cry1Ab gene from Bacillus thuringensis and neurotoxins from 61 scorpion venom have been incorporated into the baculovirus genome (8, 9). In addition, the egt 62 gene (Ac15), involved in the inhibition of the molting, has been deleted from the virus 63 significantly improving the speed of killing of the virus (10). Moreover, different attempt has 64 been conducted to improve the heterologous protein expression in the baculovirus expression 65 system (BEVS). It has been reported that simultaneous deletion of non-essential genes for the in 66 produce insertions or deletions (indels), or homology-directed repair (HDR) in presence of a 87 donor template DNA with homology to the sequence flanking the DSB (16-18). Recent 88 developments in this technology have made possible to generated precise modifications into a 89 5 wide variety of viral genomes (19). However, the CRISPR/Cas9 system has not been applied for 90 the edition of baculoviral genomes. 91In this study, we developed for first time a CRISPR/Cas9-assisted method to edit AcMNPV 92 genome in multiple ways and with different purposes. We have shown that the delivering of 93 Cas9-sgRNA ribonucleoprotein (RNP) complex through lipofection in insect cells might be 94 efficient to generate gene knock-out and knock-in. To evaluate potential app...

Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome

“…The bacmid with the deletion of all of the five nonessential genes is now commercially available (flashBACULTRA, Oxford Expression Technologies). In addition, knockout of ODV-E26 has been shown to be able to effectively increase recombinant protein production in insect larvae (Pazmiño-Ibarra et al, 2019).…”