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

Abstract

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 beyond1 and is especially suited for non-model organisms2. Effective delivery of this system, however, remains a challenge for in vivo genetic manipulation of specific tissues3, particularly the brain4, and in adult indivuduals5,6. We designed a new CRISPR-Cas9 plasmid that was inserted into a baculovirus vector to knockdown the octopamine beta subtype 2 receptor (AmOct{beta}2R), 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 AmOct{beta}2R 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 AmOct{beta}2R 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 vectors7. The newly characterized AmOct{beta}2R8 can now be assigned a functional role and other targets for gene editing are now possible using this CRISPR-Cas9 system. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/624635v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@9fba0eorg.highwire.dtl.DTLVardef@7025dforg.highwire.dtl.DTLVardef@c6706borg.highwire.dtl.DTLVardef@1fea727_HPS_FORMAT_FIGEXP M_FIG C_FIG