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

VERONICA SALOMÉ PAZMIÑO-IBARA; ADRIÀ MENGUAL-MARTÍ; ALEXANDRA MARISA TARGOVNIK; SALVADOR HERRERO

Congreso; SIP/IOBC; 2019

The CRISPR (Clustered Regularly Interspaced Short Palindromic repeats)system associated Cas9 endonuclease is a molecular tool thatenables specific sequence edition with high efficiency. The edition usingCRISPR/Cas9 system has been successfully reported in small and largeviral genomes. In this study, we have explored the use of CRISPR/Cas9system for the edition of the baculovirus genome. Guide RNAs (sgRNAs)were designed to direct a cleavage of Cas9 to different loci of the AcMNPVgenome. We showed that the delivering of Cas9-sgRNA ribonucleoprotein (RNP) complex into Sf21 insect cells through lipofection mightbe efficient to generate Indel mutations. To evaluate potential applicationof our CRISPR/Cas9 method to improve baculovirus as protein expressionvector and as biopesticide, we attempted to knock-out several genesfrom a recombinant AcMNPV form used in the baculovirus expressionsystem as well as in a natural occurring viral isolate from the same virus.Sequencing analysis revealed that the edition efficiency and the type ofchanges was variable. Depending on the targeted gene, the rate of editionranged from 10% to 40%. We analyzed the effect of the mutationsin vivo and showed, for certain mutants, improvement of baculovirus asprotein expression vector and as biopesticide. This study established thefirst evidences about the potential of CRISPR/Cas9 edition of baculovirusthat could contribute to the generation of mutants with biotechnologicalapplications.