New germline Cas9 promoters show improved performance for homing gene drive

Abstract

AbstractGene drive systems could be a viable strategy to prevent pathogen transmission or suppress vector populations by propagating drive alleles with super-Mendelian inheritance. CRISPR-based homing gene drives, perhaps the most powerful gene drive strategy, convert wild type alleles into drive alleles in heterozygotes with the help of Cas9 and gRNA. However, achieving successful outcomes with these drives often requires high performance. Specifically, it is desirable to identify Cas9 promoters that yield high drive conversion rates, minimize the formation rate of resistance alleles in both the germline and the early embryo, and limit somatic Cas9 expression. Thus far, high-performance promoters have only been discovered inAnophelesspecies. InDrosophila, thenanospromoter avoids leaky somatic expression, but at the cost of high embryo resistance from maternally deposited Cas9. To improve drive efficiency, we tested elevenDrosophila melanogastergermline promoters in several configurations. Some of the new promoters achieved higher drive conversion efficiency with minimal embryo resistance, but none could completely avoid somatic expression likenanos. However, such somatic expression often did not carry detectable fitness costs when the promoter-Cas9 elements supported a rescue homing drive targeting a haplolethal gene, suggesting somatic drive conversion. Based on our findings, we selected two Cas9 promoter lines for cage experiments with a 4-gRNA suppression drive. While one promoter exhibited substantial somatic effects, leading to a low drive equilibrium frequency, the other outperformednanos, resulting in the successful suppression of the cage population. Overall, these novel Cas9 promoters hold potential advantages for homing drives inDrosophilaspecies and may also possess valuable homologs in other organisms.