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.
The occlusion bodies (OBs) of certain alphabaculoviruses are polyhedrin-rich structures that mediate the collective transmission of tens of viral particles to the same insect host. In addition, in multiple nucleopolyhedroviruses, occlusion-derived virions (ODVs) form nucleocapsid aggregates that are delivered to the same host cell. It has been suggested that, by favoring coinfection, this transmission mode promotes evolutionarily stable interactions between different baculovirus variants. To quantify the joint transmission of different variants, we obtained OBs from cells coinfected with two viral constructs, each encoding a different fluorescent reporter, and used them for inoculating Spodoptera exigua larvae. The microscopy analysis of midguts revealed that the two reporter genes were typically segregated into different infection foci, suggesting that ODVs show limited ability to promote the co-transmission of different virus variants to the same host cell. However, a polyhedrin-deficient mutant underwent inter-host transmission by exploiting the OBs of a fully functional virus and re-acquired the lost gene through recombination, demonstrating cellular coinfection. Our results suggest that viral spatial segregation during transmission and primary infection limits interactions between different baculovirus variants, but that these interactions still occur within the cells of infected insects later in infection.
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...
Baculovirus occlusion bodies (OBs) are polyhedrin-rich structures that mediate the collective transmission of tens of viral particles to the same insect host. In addition, in multiple nucleopolyhedroviruses, occlusion-derived viruses (ODVs) form nucleocapsid aggregates that are delivered to the same host cell. It has been suggested that, by favoring coinfection, this transmission mode promotes evolutionarily stable interactions between different baculovirus variants. To investigate this, we obtained OBs from cells coinfected with two viral constructs, each encoding a different fluorescent reporter, and used them for inoculating Spodoptera exigua larvae. Microscopy analysis of midguts revealed that the two reporter genes were typically segregated into different infection foci, suggesting that ODVs do not promote the coinfection of cells with different baculovirus genetic variants. However, a polyhedrin-deficient mutant underwent inter-host transmission by exploiting the OBs of a fully-functional virus and re-acquired the lost gene through recombination, demonstrating cellular coinfection. Our results suggest that viral spatial segregation during transmission and primary infection limits interactions between different baculovirus variants, but that these interactions still occur episodically.
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