Optimized gene editing technology for <i>Drosophila melanogaster</i> using germ line-specific Cas9

TLDR

The study develops a Cas9/sgRNA method that specifically targets Drosophila germ‑line cells to produce heritable mutant alleles. The authors generated germ‑line Cas9 transgenic lines, compared promoter and sgRNA scaffold variants for mutagenesis efficiency, assessed off‑target effects, and built a web‑based database of predicted sgRNAs for fly genome engineering. The optimized system achieved a 74.2 % mutagenesis rate, proved efficient, specific, and cost‑effective, and is suitable for semi‑high‑throughput applications.

Abstract

Significance Using the recently introduced Cas9/sgRNA technique, we have developed a method for specifically targeting Drosophila germ-line cells to generate heritable mutant alleles. We have established transgenic lines that stably express Cas9 in the germ line and compared different promoters and scaffolds of sgRNA in terms of their efficiency of mutagenesis. An overall mutagenesis rate of 74.2% was achieved with this optimized system, as determined by the number of mutant progeny out of all progeny screened. We also evaluated the off-targets associated with the method and established a Web-based resource, as well as a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Our results demonstrate that this optimized Cas9/sgRNA system in Drosophila is efficient, specific, and cost-effective and can be readily applied in a semi-high-throughput manner.