Inherently confinable split-drive systems in Drosophila

Abstract

CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.

Gene drives gaining speed

“…In contrast to suppression drives, the impediment to drive success imposed by functional alleles resistant to Cas9 cleavage is much less of a concern for modification drives, which are designed not to impose any significant fitness costs 70 . Consistent with the expectation that rare functional drive-resistant alleles should have little, if any, advantage over a fitness-neutral drive allele, such variants have not been observed to expand in small population cages 54 , 57 , 85 , in contrast to the strong positive selection imposed on this class of alleles by suppression drives 47 , 86 . These comparative considerations notwithstanding, it still makes good engineering sense for modification drives to achieve maximal introduction into a target population.…”

“…A broadly transferable system for eliminating non-functional drive-resistant alleles, present either as sequence variants in a population 78 , 92 – 95 or arising during the gene-drive process 45 – 47 , 49 , 60 , 76 , 77 , is to insert the drive into an essential gene and endow it with a recoded cDNA-restoring gene function 53 – 57 , 61 . Such recoded-drive elements can survive when homozygous.…”

“…However, non-functional alleles resulting from non-homologous end-joining (NHEJ) will be eliminated by a combination of dominantly acting lethal or sterile mosaicism (see below) and standard negative selection. Furthermore, if one chooses a functionally critical site in a target gene, such as a catalytic centre in an essential enzyme 54 , 56 , 57 or in a signal required for cellular trafficking such as a membrane tethering motif 57 , the likelihood of potentially competing functional NHEJ alleles arising is greatly diminished.…”

“…The second, very potent effect, is that mothers passing on a non-functional mutant allele of the target gene to progeny also transmit Cas9–guide RNA complexes maternally. If the essential gene is broadly active in the organism (for example, a ubiquitously expressed enzyme), then the paternal allele in those progeny can be mutagenized in a mosaic fashion by those complexes, leading to a pseudo homozygous phenotype and hence lethality or sterility depending on the nature of the targeted essential gene 54 , 56 , 57 . We have referred to this dominantly acting phenomenon as lethal (or sterile) mosaicism, which was discovered in the context of developing an allelic-drive system 60 , where this process plays an essential role, acting like a catalytic filter to eliminate individuals inheriting loss-of-function copying errors.…”

“…These drive elements can be used to reduce mosquito populations (suppression) or to render them incapable of transmitting pathogens (modification; for example, by including cargo genes encoding anti-malarial effectors 46 ). For modification systems, incorporating functional recoded versions of genes into drives can greatly improve their performance 54 – 57 .…”