Highly Improved Gene Targeting by Germline-Specific Cas9 Expression in <i>Drosophila</i>

TLDR

The authors present a simple, highly efficient, and scalable Cas9-based platform for systematic gene targeting in Drosophila. The system uses a germline‑specific nanos‑promoter Cas9 strain crossed with a ubiquitously expressed gRNA strain to generate active Cas9–gRNA complexes that cleave target sites in germ cells. Using this platform, the authors rapidly produced mutants in seven neuropeptide and two microRNA genes, achieved 60 % germline transmission of mutations, and induced internal deletions at 4.3–23 % frequency with dual gRNAs, markedly improving efficiency over previous transient Cas9 methods.

Abstract

Abstract We report a simple yet extremely efficient platform for systematic gene targeting by the RNA-guided endonuclease Cas9 in Drosophila. The system comprises two transgenic strains: one expressing Cas9 protein from the germline-specific nanos promoter and the other ubiquitously expressing a custom guide RNA (gRNA) that targets a unique site in the genome. The two strains are crossed to form an active Cas9–gRNA complex specifically in germ cells, which cleaves and mutates the target site. We demonstrate rapid generation of mutants in seven neuropeptide and two microRNA genes in which no mutants have been described. Founder animals stably expressing Cas9–gRNA transmitted germline mutations to an average of 60% of their progeny, a dramatic improvement in efficiency over the previous methods based on transient Cas9 expression. Simultaneous cleavage of two sites by co-expression of two gRNAs efficiently induced internal deletion with frequencies of 4.3–23%. Our method is readily scalable to high-throughput gene targeting, thereby accelerating comprehensive functional annotation of the Drosophila genome.