Successful delivery of CRISPR-Cas9 with a baculovirus vector for insect brain targets

CRISPR-Cas9 (clustered, regularly interspaced, short palindromic repeats with CRISPR-associated protein 9) is a powerful, versatile, and cost-effective molecular tool that can be used for genetic engineering purposes and beyond ¹ and is especially suited for non-model organisms ² . Effective delivery of this system, however, remains a challenge for in vivo genetic manipulation of specific tissues ³ , particularly the brain ⁴ , and in adult indivuduals ^5,6 . We designed a new CRISPR-Cas9 plasmid that was inserted into a baculovirus vector to knockdown the octopamine beta subtype 2 receptor ( AmOctβ2R ), a transmembrane protein found in the mushroom body neurons of the honey bee ( Apis mellifera ) brain, to determine if octopamine plays a role in appetite regulation. We first confirmed that gene editing of AmOctβ2R is possible with Sanger sequencing. We then demonstrated expression of the CRISPR-Cas9 system with the baculovirus vector in vitro using live cell imaging, flow cytometry analysis, and in vivo using confocal imaging, showing widespread expression in the cells and throughout the honey bee brain, three days post treatment. There was also in vitro and in vivo knockdown of AmOctβ2R three days post-infection, that corresponded with appetite suppression in starved forager bees. Our findings suggest that we successfully delivered the CRISPR-Cas9 system and knocked down AmOctB2R in neuronal cells of the honey bee brain that were previously inaccessible due to the blood brain barrier and lack of infectivity of lentivirus vectors ⁷ . The newly characterized AmOctβ2R ⁸ can now be assigned a functional role and other targets for gene editing are now possible using this CRISPR-Cas9 system.