Abstract
CRISPR-Cas9 nuclease-based gene drives rely on inducing chromosomal breaks in the germline that are repaired in ways that lead to a biased inheritance of the drive. Gene drives designed to impair female fertility can suppress populations of the mosquito vector of malaria. However, strong unintended fitness costs, due to ectopic nuclease expression, and high levels of resistant mutations, limited the potential of the first generation of gene drives to spread.
Here we show that changes to regulatory sequences in the drive element, designed to contain nuclease expression to the germline, confer improved fecundity over previous versions and generate drastically lower rates of target site resistance. We employed a genetic screen to show that this effect is explained by reduced rates of end-joining repair of DNA breaks at the target site caused by deposited nuclease in the embryo.
Highlighting the impact of deposited Cas9, many of the mutations arising from this source of nuclease activity in the embryo are heritable, thereby having the potential to generate resistant target sites that reduce the penetrance of the gene drive.
Finally, in cage invasion experiments these gene drives show improved invasion dynamics compared to first generation drives, resulting in greater than 90% suppression of the reproductive output and a delay in the emergence of target site resistance, even at a resistance-prone target sequence. We shed light on the dynamics of generation and selection of resistant alleles in a population by tracking, longitudinally, the frequency of resistant alleles in the face of an invading gene drive. Our results illustrate important considerations for future gene drive design and should expedite the development of gene drives robust to resistance.
Footnotes
Some of the main text has been modified to improve flow. Substantial changes are: -the addition of modelling of gene drive performance in invasion experiments -changes to the previous figure 3 (repair outcomes of deposited Cas9 nuclease). The results in this figure were previously compromised by a technical artefact that occurred due to the binding site of one of the primers, used to prepare pooled amplicons for sequencing, being polymorphic in the mosquito colony. The resulting differential efficiency of the PCR on certain haplotypes partially confounded accurate determination of the relative representation of alleles in the pooled sequence. This was rectified by the use of primers that bound non-polymorphic and all analyses were repeated. These are now included in the updated figure. Additionally we continued this experiment into the F3 to look at the heritability of mutations caused in the f2 generation by the deposited nuclease. - a new figure detailing meiotic end-joining following Cas9 activity