Fitness effects of CRISPR endonucleases in Drosophila melanogaster populations

Abstract

CRISPR/Cas9 provides a highly efficient and flexible genome editing technology with numerous potential applications ranging from gene therapy to population control. Some proposed applications involve the integration of CRISPR/Cas9 endonucleases into an organism's genome, which raises questions about potentially harmful effects to the transgenic individuals. One example for which this is particularly relevant are CRISPR-based gene drives conceived for the genetic alteration of entire populations. The performance of such drives can strongly depend on fitness costs experienced by drive carriers, yet relatively little is known about the magnitude and causes of these costs. Here, we assess the fitness effects of genomic CRISPR/Cas9 expression in Drosophila melanogaster cage populations by tracking allele frequencies of four different transgenic constructs that allow us to disentangle 'direct' fitness costs due to the integration, expression, and target-site activity of Cas9, from fitness costs due to potential off-target cleavage. Using a maximum likelihood framework, we find that a model with no direct fitness costs but moderate costs due to off-target effects fits our cage data best. Consistent with this, we do not observe fitness costs for a construct with Cas9HF1, a high-fidelity version of Cas9. We further demonstrate that using Cas9HF1 instead of standard Cas9 in a homing drive achieves similar drive conversion efficiency. These results suggest that gene drives should be designed with high-fidelity endonucleases and may have implications for other applications that involve genomic integration of CRISPR endonucleases.

The suppressive potential of a gene drive in populations of invasive social wasps is currently limited

“…We also do not model early or late germline resistance allele formation that could happen due to asynchronous Cas9 expression and homology directed repair 24 , 70 , 76 . Finally, the gene drive fitness cost is implemented as being fully dominant, whereas the mechanism could be more complex 63 . All in all, we see that a gene drive already has a very limited set of parameters under which it could suppress a population of social wasps.…”

“…We evaluate the sensitivity of results to the default probability of cutting, probability of non-homologous end-joining, and probability of functional repair. We also model a fitness cost of carrying the gene drive abstracted as a certain probability of mortality before mating ( , default 0 63 ), which we also evaluate in the sensitivity analysis. We conservatively assume that the fitness cost of carrying the gene drive has complete dominance, that is, it is equally deleterious for homo-, hemi-, and heterozygotes.…”

Anopheles homing suppression drive candidates exhibit unexpected performance differences in simulations with spatial structure

“…In conclusion, our modeling indicates that several promising candidates for suppression gene drives could have substantially different overall effectiveness in spatial mosquito populations than predicted by panmictic population models. Specifically, we believe that a standard homing suppression drive using the nos promoter ( Carballar-Lejarazú et al, 2020 ; Hammond et al, 2021 ) for Cas9 (possibly a high fidelity variant to avoid off-target cleavage Langmüller et al, 2022 ) and targeting dsx ( Simoni et al, 2020 ; Kyrou et al, 2018 ) with two or more closely spaced gRNA target sites ( Yang et al, 2022 ; Champer et al, 2020b ) may be the optimal combination of currently available tools for population suppression of Anopheles mosquitoes.…”

Performance characteristics allow for confinement of a CRISPR toxin-antidote gene drive designed for population suppression

“…While this represents only a modest fitness cost, note that it is a per-allele (multiplicative) fitness cost for both heterozygotes and homozygotes. A TADE suppression drive would not have a costly cargo gene, and the fitness of drive alleles without cargo would usually be close to wild-type 46 . Rescue efficiency of the haplolethal target could also likely be highly effective, preventing significant fitness costs 47 .…”