Resistance is futile: A CRISPR homing gene drive targeting a haplolethal gene

Abstract

Engineered gene drives are being explored as a potential strategy for the control of vector-borne diseases due to their ability to rapidly spread genetic modifications through a population. While an effective CRISPR homing gene drive for population suppression has recently been demonstrated in mosquitoes, formation of resistance alleles that prevent Cas9 cleavage remains the major obstacle for drive strategies aiming at population modification, rather than elimination. Here, we present a homing drive in Drosophila melanogaster that reduces resistance allele formation below detectable levels by targeting a haplolethal gene with two gRNAs while also providing a rescue allele. This is because any resistance alleles that form by end-joining repair will typically disrupt the haplolethal target gene, rendering the individuals carrying them nonviable. We demonstrate that our drive is highly efficient, with 91% of the progeny of drive heterozygotes inheriting the drive allele and with no resistance alleles observed in the remainder. In a large cage experiment, the drive allele successfully spread to all individuals. These results show that a haplolethal homing drive can be a highly effective tool for population modification.

Computational and experimental performance of CRISPR homing gene drive strategies with multiplexed gRNAs

“…Resistance alleles that disrupt the function of the target gene incur large fitness costs in several drive designs, which would make resistance substantially less likely to block the spread of the drive. However, two studies utilizing two gRNAs 13,16 showed somewhat lower increases in efficiency than that predicted by simple models of multiple gRNAs [22][23][24] . This is partially because most models assume that cleavage and repair by either homology-directed repair or end-joining occur sequentially at each gRNA target site.…”

“…In our model, we assume no special targeting of conserved sequences and thus estimate that resistance sequences that preserve the function of the target gene form in 10% of cases 12,13 . In reality, this could be substantially reduced 13,16,21 . In our model, we further assume that a resistance sequence preserving the function of the target gene must form independently at each gRNA target site for the target gene to retain its function.…”

“…For this drive, females are rendered sterile unless they possess at least one wild-type allele or resistance allele that preserves the function of the target gene. We also consider approaches for population modification that target a haplolethal or recessive lethal gene, where the drive has a recoded sequence of the gene that cannot be cleaved by the drive's gRNAs 16 . In the haplolethal approach, any individual with a resistance allele that disrupts the target gene is nonviable, removing these alleles from the population.…”

“…The drive is then copied into the wild-type site via homology-directed repair, increasing the frequency of the drive allele in the population. Thus far, CRISPR homing gene drives have been demonstrated in yeast [5][6][7][8] , flies [9][10][11][12][13][14][15][16] , mosquitoes [17][18][19] , and mice 20 .…”

“…While formation of resistance alleles remains the primary obstacle to construction of efficient gene drives, substantial progress has been made toward overcoming this challenge. For example, a suppression type drive in Anopheles gambiae 21 and a modification type drive in Drosophila melanogaster 16 avoided issues with resistance alleles by targeting an essential gene. Because of this, resistance alleles that disrupted the function of the target gene had substantially lower fitness than the drive.…”