Systematic Evaluation of <i>Drosophila</i> CRISPR Tools Reveals Safe and Robust Alternatives to Autonomous Gene Drives in Basic Research

TLDR

CRISPR/Cas technology enables rapid, site‑specific genome modification across many organisms, yet in *Drosophila melanogaster* diverse tools have produced uncertainty about practical use and the proportion of target sites that can be edited efficiently. This study systematically evaluates available CRISPR/Cas reagents and methods in *Drosophila* to resolve these uncertainties. The authors performed gene editing at many target sites using a highly active Cas9 line together with a library of transgenic gRNA strains. They found that most target sites can be mutated with high efficiency, that evidence‑based choices of Cas9 sources and knock‑in strategies can be made, and that optimized CRISPR with independent transgenes is as efficient, more versatile, and safer than autonomous gene‑drive approaches.

Abstract

Abstract The Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated (CRISPR/Cas) technology allows rapid, site-specific genome modification in a wide variety of organisms . Proof-of-principle studies in Drosophila melanogaster have used various CRISPR/Cas tools and experimental designs, leading to significant uncertainty in the community about how to put this technology into practice. Moreover, it is unclear what proportion of genomic target sites can be modified with high efficiency. Here, we address these issues by systematically evaluating available CRISPR/Cas reagents and methods in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for somatic and germline genome engineering and conclude that optimized CRISPR with independent transgenes is as efficient, more versatile, and does not represent a biosafety risk.