In a recent PNAS article, Champer et al. report variable rates of gene drive-based resistance allele formation.
Gene drives have shown early promise as the next big thing in controlling insects both for the benefit of agricultural production and reducing insect-borne disease transmission. The power of this system lies in its ability to convert heterozygous progeny into homozygotes for the gene drive allele by endogenous Cas9-editing.
Over time, alleles resistant to drive recognition evolve throughout the population, leading to the ultimate decay of the gene drive. Champer et al describe two types of resistance alleles, “r1” and “r2″, in their Drosophila melanogaster system. Non-homologous end joining repair strategies lead to both types of alleles, where r2 alleles produce dysfunctional copies of the target gene. r1 alleles remain functional copies, but unrecognizable to the gRNA-Cas9 complex.
In this report, Champer et. al uses three separate strategies to reduce the formation of resistance alleles: guide RNA multiplexing — wherein multiple guides target the same gene at different sites, the use of paternally deposited Cas9 with an autosomal drive, and modifying expression through promoter sequences, vasa and nanos.
The multiplexing approach wherein multiple gRNA sequences target the same target gene effectively reduces r2 resistance allele formation by about 17%. However, while the single-guide approach allows only for the production of r1 or r2 resistance alleles, the two-guide system can create in combination of +, r1, and r2 alleles at the two target sites. Multiplexing may not wholly solve the issue, but drive transmission appears more efficient with 2 gRNA constructs.
One contributor to resistance allele formation involves excess maternally deposited Cas9. Sperm cells because of their reduced cytoplasmic volume will transmit less Cas9 to the embryo that was expressed during gametogenesis. They suggest that if drive conversion could be restricted exclusively to male germline, it would be possible to reduce embryonic resistance formation.
The promoter under which the gene drive expresses also plays a key role in the formation of resistance alleles. Champer et al. compare vasa and nanos promoters in their drive flies. The group notes that resistance alleles can also arise in nongermline cells due to somatic Cas9 transcription. They demonstrate that nanos lacks the somatic expression seen with vasa, which may be vital to the dissemination of a gene drive in which gene disruption may come with a fitness cost.
This report expands significantly on our understanding of resistance allele formation in drives and highly suggests that guide multiplexing is the most versatile and promising approach to reduce drive resistance overall. Another key point to consider remains that resistance rates are highly dependent upon the target gene itself, seeking alternate targets may prove necessary in some scenarios.