Base editing using CRISPR/Cas9 in Drosophila

Abstract

Cas9 and a guide RNA function to target specific genomic loci for generation of a double stranded break. Catalytic dead versions of Cas9 (dCas9) no longer cause double stranded breaks and instead can serve as molecular scaffolds to target additional enzymatic proteins to specific genomic loci. To generate mutations in selected genomic residues, dCas9 can be used for genomic base editing by fusing a cytidine deaminase to induce C>T (or G>A) mutations at targeted sites. Here, we test base editing in Drosophila by expressing a transgenic Drosophila base editor (DBE2, based on the mammalian BE2) which consists of a fusion protein of cytidine deaminase, dCas9, and uracil glycosylase inhibitor. We utilized transgenic lines expressing gRNAs along with pan-tissue expression of the Drosophila Base Editor (Actin5C-DBE2) and found high rates of base editing at multiple targeted loci in the 20 bp target sequence. Highest rates of conversion of C>T were found in positions 3-9 of the gRNA targeted site, with conversion reaching nearly 100% of targeted DNA is somatic tissues. The simultaneous use of two gRNA targeting a genomic region spaced [~]50 bps apart led to mutations between the two gRNA targets, implicating a method to broaden the available sites accessible to targeting. These results indicate base editing is efficient in Drosophila, and could be used to induce point mutations at select loci.