Courtship assays.

<div><p>Here, we demonstrate that single strand annealing (SSA) can be co-opted for the precise autocatalytic excision of a drive element. We have termed this technology <u>Re</u>peat <u>M</u>ediated <u>E</u>xcision of a <u>D</u>rive <u>E</u>lement (ReMEDE). By engineering direct repeats flanking the drive allele and inducing a double-strand DNA break (DSB) at a second endonuclease target site within the allele, we increased the utilization of SSA repair. ReMEDE was incorporated into the mutagenic chain reaction (MCR) gene drive targeting the <i>yellow</i> gene of <i>Drosophila melanogaster</i>, successfully replacing drive alleles with wild-type alleles. Sequencing across the Cas9 target site confirmed transgene excision by SSA after pair-mated outcrosses with yReMEDE females, revealing ~4% inheritance of an engineered silent TcG marker sequence. However, phenotypically wild-type flies with alleles of indeterminate biogenesis also were observed, retaining the TGG sequence (~16%) or harboring a silent gGG mutation (~0.5%) at the PAM site. Additionally, ~14% of alleles in the F2 flies were intact or uncut paternally inherited alleles, indicating limited maternal deposition of Cas9 RNP. Although ReMEDE requires further research and development, the technology has some promising features as a gene drive mitigation strategy, notably its potential to restore wild-type populations without additional transgenic releases or large-scale environmental modifications.</p></div>