Inherently confinable split-drive systems in Drosophila

TLDR

CRISPR‑based gene‑drive systems copy via HDR but are impeded by NHEJ‑generated resistant alleles that can halt drive spread. The authors hypothesize that targeting essential genes with recoded sequences will combine maternal NHEJ clearance and recessive culling to enhance drive performance. They test split gene‑drive constructs inserted into essential viability genes rab5, rab11, prosalpha2 and the fertility gene spo11 in *Drosophila melanogaster*. Crosses show variable, sex‑biased transmission, while cage trials reveal distinct trajectories driven by chromosome damage or lethal/sterile mosaic phenotypes, resulting in inherently confinable drives.

Abstract

Abstract CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability ( rab5 , rab11 , prosalpha2 ) or fertility ( spo11 ). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.