Fitness effects of CRISPR endonucleases inDrosophila melanogasterpopulations

Abstract

CRISPR/Cas9 systems provide a highly efficient and flexible genome editing technology with numerous potential applications in areas ranging from gene therapy to population control. Some proposed applications involve CRISPR/Cas9 endonucleases integrated into an organisms genome, which raises questions about potentially harmful effects to the transgenic individuals. One application where this is particularly relevant are CRISPR-based gene drives, which promise a mechanism for rapid 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, designed to disentangle direct fitness costs due to the integration, expression, and target-site activity of Cas9 from costs due to potential off-target cleavage. Using a maximum likelihood framework, we find a moderate level of fitness costs due to off-target effects but do not detect significant direct costs. Costs of off-target effects are minimized 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. Our 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.

A homing suppression gene drive with multiplexed gRNAs maintains high drive conversion efficiency and avoids functional resistance alleles

“…These issues could potentially be addressed by changing the target gene to one of many other possible female fertility genes that does not have a germline-related maternal effect. Another possibility is off-target cleavage effects as seen previously 53 , which would likely be exacerbated by multiplexed gRNAs. However, such an issue could be addressed relatively easily by using high fidelity Cas9 nucleases that show little to no off-target cleavage [59][60][61][62][63][64][65] , which have been shown to have similar drive performance 53 .…”

“…Another possibility is off-target cleavage effects as seen previously 53 , which would likely be exacerbated by multiplexed gRNAs. However, such an issue could be addressed relatively easily by using high fidelity Cas9 nucleases that show little to no off-target cleavage [59][60][61][62][63][64][65] , which have been shown to have similar drive performance 53 . By contrast, if fitness costs are caused by the expression of the drive components themselves, they may be more difficult to directly address.…”

“…To quantify drive fitness costs, we modified a previously developed maximum likelihood inference framework 25,52 . Similar to a previous study 53 , we extended the model to two unlinked loci (drive site and a site representing undesired mutations from off-target cleavage that impose a fitness cost). The Maximum Likelihood inference framework is implemented in R (v. 4.0.3) 54 and is available on GitHub (https://github.com/MesserLab/HomingSuppressionDrive).…”

“…We set the germline cut rate at the off-target locus to 1 and did not model additional off-target cuts in embryos with drive-carrying mothers. This represents the simplest model of mostly distant off-target sites that are mostly cut in the germline when Cas9 cleavage rates are highest (actual off-target cleavage would likely be at many sites at much lower rates, with some linked to the drive alleles, which would not be possible to easily model with our maximum likelihood method 53 ). We assumed that in drive carriers at the beginning of the experiment, 50% of the off-target sites are cut because the drive carrier flies all came from male drive heterozygotes.…”

Modelling homing suppression gene drive in haplodiploid organisms

“…It affects female fecundity directly, and fitness also proportionally reduces the chance that a male will be selected as a mate. Our default fitness value was 0.95, indicating a high quality but imperfect drive 50 . Negative fitness effects can also stem from somatic expression in female drive/wild-type heterozygotes, which can disrupt wild-type alleles in somatic cells that are necessary for female reproduction 5,27 .…”