CRISPR/Cas9 mediated genome engineering in Drosophila

• Targeted mutagenesis of Drosophila genes with CRISPR/Cas9 nucleases.
• Rapid production of novel synthetic guide RNAs by PCR and in vitro transcription.
• Simple and highly sensitive detection of mutations by high resolution melt analysis.
• Efficient generation of indels that can be transmitted through the germline.

Genome engineering has revolutionised genetic analysis in many organisms. Here we describe a simple and efficient technique to generate and detect novel mutations in desired target genes in Drosophila melanogaster. We target double strand breaks to specific sites within the genome by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed synthetic guide RNA into Drosophila embryos. The small insertion and deletion mutations that result from inefficient non-homologous end joining at this site are detected by high resolution melt analysis of whole flies and individual wings, allowing stable lines to be made within 1 month.

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