CRISPR-/Cas9-Mediated Precise and Efficient Genome Editing in Drosophila

Abstract

The CRISPR/Cas9 system provides the means to make precise and purposeful modifications to the genome via homology-directed repair (HDR). In Drosophila, a wide variety of tools provide flexibility to achieve these ends. Here, we detail a method to generate precise genome edits via HDR that is efficient and broadly applicable to any Drosophila stock or species. sgRNAs are first tested for their cleavage efficiency by injecting embryos with Cas9/sgRNA ribonucleoproteins using commercially available Cas9 protein. Using an empirically validated sgRNA, HDR is performed using a donor repair plasmid that carries two transformation markers. A fluorescent eye marker that can be seamlessly removed using PiggyBac transposase marks integration of the repair sequence. A counter-selection marker that produces small rough eyes via RNAi against eyes absent is used to screen against imprecise HDR events. Altogether, the enhancements implemented in this method expand the ease and scope of achieving precise CRISPR/Cas9 genome edits in Drosophila.

Keywords: CRISPR; Drosophila; Genome editing; Germline; Homology-directed repair.

Fig. 1

Workflow to produce precise genome edits in Drosophila using CRISPR/Cas9. Multiple sgRNAs are designed, and Cas9/sgRNA RNPs are screened in Drosophila embryos to ensure efficient sgRNA cleavage activity in vivo. A donor repair plasmid is then computationally designed and constructed via Gibson Assembly. The Cas9/sgRNA RNP and donor repair plasmid are then injected together into Drosophila embryos and screened for successful transformation via HDR using a DsRed eye marker. This marker is then removed using a PiggyBac transposase without leaving scars in the genome sequence

Fig. 2

In vivo screening of sgRNA cleavage activity. PCR products (826bp in size) of an sgRNA target site in the lncRNA CR45715 gene from both uninjected and RNP-injected embryos are digested by T7EI. Three representative embryos (from eight total) are shown for each. Predicted T7EI cleavage products are 596 and 230 bp. The 596-bp product (arrowhead) is clearly visible in two RNP-injected embryos, though the 230-bp product is likely too faint to visualize. Note that T7EI produces digestion products at ~500 bp and ~300 bp even in uninjected embryos, but these are easily distinguished from the sgRNA-induced cleavage products. This sgRNA was subsequently used to generate multiple edits in the region via HDR

Fig. 3

Design of the sgRNA and donor repair plasmid. Important design considerations are shown at and around the sgRNA target site. The NGG PAM site is adjacent to the sgRNA target site in the genome, but is not included in the sgRNA sequence itself. A nearby TTAA sequence in the genome (ideally within 30bp of double-stranded break site) serves as site of insertion for the scarless DsRed cassette. This TTAA site can also be located within the sgRNA target sequence or within the novel insert sequence itself. The sgRNA target site in the donor repair plasmid is inactivated by placing the novel insert sequence between the 20bp sgRNA target site and the PAM site. This can also be achieved by mutating the PAM site or the very 3′ end of the sgRNA target site

Fig. 4

Identification of precise HDR edits by eye morphology and fluorescence. Successful HDR edits using this protocol are evidenced by flies with wild-type eye morphology and DsRed fluorescence (A, A′). The fluorescent DsRed eye marker is then removed from the genome using a PiggyBac transposase, leaving non-fluorescent eyes with wild-type morphology (B, B′). An imprecise HDR edit that integrates the entire donor repair plasmid into the genome is evidenced by the DsRed eye marker and expression of the GMR>eya(RNAi) transgene, which produces a rough small eye phenotype (C, C′)