Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila

Abstract

The CRISPR-associated RNA-guided nuclease Cas9 has emerged as a powerful tool for genome engineering in a variety of organisms. To achieve efficient gene targeting rates in Drosophila, current approaches require either injection of in vitro transcribed RNAs or injection into transgenic Cas9-expressing embryos. We report a simple and versatile alternative method for CRISPR-mediated genome editing in Drosophila using bicistronic Cas9/sgRNA expression vectors. Gene targeting with this single-plasmid injection approach is as efficient as in transgenic nanos-Cas9 embryos and allows the isolation of targeted knock-out and knock-in alleles by molecular screening within 2 months. Our strategy is independent of genetic background and does not require prior establishment of transgenic flies.

Keywords: CRISPR; Cas9; Drosophila; HDR.

Figure 1

A bicistronic Drosophila CRISPR/Cas9 vector. (A) Schematic map of pDCC6, with the sgRNA cassette under the control of the Drosophila U6:96Ab (U6-2) promoter as well as an hsp70Bb promoter driving Cas9 expression. (B) gRNA sequences are inserted between the U6 promoter and the sgRNA scaffold via two BbsI sites. (C) gRNAs are cloned as complementary oligonucleotide pairs with suitable overhangs and an additional G (bold, required for RNA PolIII transcription) preceding the target-specific 20 nt protospacer (gray).