Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance

Abstract

Homing-based gene drives use a germline source of nuclease to copy themselves at specific target sites in a genome and bias their inheritance. Such gene drives can be designed to spread and deliberately suppress populations of malaria mosquitoes by impairing female fertility. However, strong unintended fitness costs of the drive and a propensity to generate resistant mutations can limit a gene drive’s potential to spread. Alternative germline regulatory sequences in the drive element confer improved fecundity of carrier individuals and reduced propensity for target site resistance. This is explained by reduced rates of end-joining repair of DNA breaks from parentally deposited nuclease in the embryo, which can produce heritable mutations that reduce gene drive penetrance. We tracked the generation and selection of resistant mutations over the course of a gene drive invasion of a population. Improved gene drives show faster invasion dynamics, increased suppressive effect and later onset of target site resistance. Our results show that regulation of nuclease expression is as important as the choice of target site when developing a robust homing-based gene drive for population suppression.

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

“…Nevertheless, the drive frequency did not decrease systematically over the course of the experiment after the initial increase of the drive, suggesting that functional r1 resistance alleles did not form at a high rate. This is in contrast to two recent studies of homing suppression drives where r1 alleles outcompeted the drive alleles despite lower population sizes and higher drive efficiency 26,34 .…”

“…Fitness costs from the drive would, in this case, further reduce the equilibrium drive frequency and resulting genetic load. Homing suppression drives in mosquitoes 27,29,34 , therefore, have demonstrated superior genetic loads compared to this drive in fruit flies. In these mosquito drives, the higher somatic fitness costs were more than compensated for by a higher drive conversion efficiency.…”

Double drives and private alleles for localised population genetic control

“…Genetic parameter values (rates of DNA cleavage, rates of alternative repair pathways, and the sex ratio produced by X-shredding) are as estimated from An. gambiae (S1 Table) [11,25,52]. Constructs may be inserted into a haplosufficient or haplo-insufficient female-essential gene (in which case gene function is disrupted), a selectively neutral sequence (in which case the insertion is also selectively neutral), or a haplo-sufficient or haplo-insufficient gene required for male and female viability (in which case the insertion is again selectively neutral, because it contains a re-coded version of the target gene [14,26,29], or is inserted in an artificial intron [30]).…”

“…gambiae mosquitoes [11,50], but may also work more broadly [51]. Note that the optimal timing of homing and X-shredding during gametogenesis may be different, requiring different and compatible control sequences, which will need to be taken into account in construct design [50,52]. The ability to control gRNA expression in a tissue-specific manner would be helpful in this regard.…”

Potential use of gene drive modified insects against disease vectors, agricultural pests and invasive species poses new challenges for risk assessment

“…In suppressive GD systems, declining population numbers reduce the frequency of potential mutation events that could lead to resistance, despite the higher selection pressure for resistance mutations compared to population modification GDs. Various strategies are currently being explored to overcome resistance evolution: multiplexing gRNA targeting different target DNAs [26,27], targeting ultra-conserved and functionally constrained genes essential for survival or fertility [28][29][30], optimising/ regulating GD expression to reduce end-joining activity [31], stacking multiple cargo/payload genes in the same host individual [32], designing engineered GDs that target conserved or haploinsufficient genes that also carry a recoded cDNA restoring endogenous gene activities [30,[33][34][35][36][37], minimizing fitness costs [38], and combining multiple engineered GD approaches [39]. While potential future GD failure (i.e.…”