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Gene drive is a genetic engineering technology that can enable super-mendelian inheritance of specific alleles, allowing them to spread through a population. New gene drive types have increased flexibility, offering options for confined modification or suppression of target populations. Among the most promising are CRISPR toxin-antidote gene drives, which disrupt essential wild-type genes by targeting them with Cas9/gRNA. This results in their removal, increasing the frequency of the drive. All these drives rely on having an effective rescue element, which consists of a recoded version of the target gene. This rescue element can be at the same site as the target gene, maximizing the chance of efficient rescue, or at a distant site, which allows useful options such as easily disrupting another essential gene or increasing confinement. Previously, we developed a homing rescue drive targeting a haplolethal gene and a toxin-antidote drive targeting a haplosufficient gene. These successful drives had functional rescue elements but suboptimal drive efficiency. Here, we attempted to construct toxin-antidote drives targeting these genes with a distant-site configuration from three loci in Drosophila melanogaster. We found that additional gRNAs increased cut rates to nearly 100%. However, all distant-site rescue elements failed for both target genes. Furthermore, one rescue element with a minimally recoded sequence was used as a template for homology-directed repair for the target gene on a different chromosomal arm, resulting in the formation of functional resistance alleles. Together, these results can inform the design of future CRISPR-based toxin-antidote gene drives.

Though engineered gene drives hold great promise for spreading through and suppressing populations of disease vectors or invasive species, complications such as resistance alleles and spatial population structure can prevent their success. Additionally, most forms of suppression drives, such as homing drives or driving Y chromosomes, will generally spread uncontrollably between populations with even small levels of migration. The previously proposed CRISPR-based toxin-antidote system called toxin-antidote dominant embryo (TADE) suppression drive could potentially address the issues of confinement and resistance. However, it is a relatively weak form of drive compared to homing drives, which might make it particularly vulnerable to spatial population structure. In this study, we investigate TADE suppression drive using individual-based simulations in a continuous spatial landscape. We find that the drive is actually more confined than in simple models without space, even in its most efficient form with low cleavage rate in embryos from maternally deposited Cas9. Furthermore, the drive performed well in continuous space scenarios if the initial release requirements were met, suppressing the population in a timely manner without being severely affected by chasing, a phenomenon in which wild-type individuals avoid the drive by recolonizing empty areas. At higher embryo cut rates, the drive loses its ability to spread, but a single, widespread release can often still induce rapid population collapse. Thus, if TADE suppression gene drives can be successfully constructed, they may play an important role in control of disease vectors and invasive species when stringent confinement to target populations is desired.