A homing suppression gene drive with multiplexed gRNAs maintains high drive conversion efficiency and avoids functional resistance alleles

Abstract

Gene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive in Drosophila melanogaster that utilized multiplexed gRNAs to inhibit the formation of functional resistance alleles in its female fertility target gene. The gRNA target sites were placed close together, preventing reduction in drive conversion efficiency. The construct reached a moderate equilibrium frequency in cage populations without apparent formation of resistance alleles. However, a moderate fitness cost prevented suppression of the cage population. Nevertheless, our results experimentally demonstrate the viability of the multiplexed gRNAs strategy in homing type suppression gene drives.

Modelling homing suppression gene drive in haplodiploid organisms

“…Fortunately, we found that the strategy of targeting a haplosufficient but essential female-specific gene with a homing drive was still able to achieve high genetic load (and though we modelled a female fertility gene, results would be broadly similar with a female viability gene). This type of gene drive has been well studied, with the latest drives even circumventing functional resistance alleles by careful target site selection 5 or multiplexed gRNAs 42 . Because haplodiploid drives are somewhat more vulnerable to such functional resistance alleles, both methods would likely also be needed to avoid functional resistance in large, natural haplodiploid populations.…”

“…However, our study indicates that homing suppression drives in haplodiploids would need to keep somatic activity and embryo resistance allele formation even lower than in diploid drives to be effective. Though a rigorous requirement, there is still some flexibility in the specific values, and previously constructed drives have already achieved low enough somatic activity in flies 42 and embryo resistance in mosquitoes 5 . Surprisingly, we found that in continuous space models, female fertility homing suppression drives perform nearly as well in haplodiploid species as in diploids.…”

“…In this study, we investigate this drive in detail, including its properties such as rate of spread and especially its result in failure of a suppression drive. However, multiple gRNAs 16,42 and conserved target sites 5 could be used to limit the formation of functional resistance alleles (perhaps at the cost of drive conversion efficiency 16,42,43 ), so we only explicitly modelled them in some of our simulations.…”

Experimental demonstration of tethered gene drive systems for confined population modification or suppression

“…A 10 mM Tris-HCl, 100 μM EDTA solution at pH 8.5 was used for the injection. Both lines containing split homing modification 21 and suppression 47 drives were generated in previous studies.…”

“…Details of this approach and its application to cage population data were previously described 48 . The fitness parameters for both homing lines used here have been previously estimated 21,47 . For the TARE line, we estimated the viability, female fecundity, and male mating success parameters and the effective population size using a model that assumes a co-dominant, multiplicative fitness effect of the TARE allele (heterozygotes were assigned fitness equal to the square root of homozygotes).…”