A functioning gene drive system could fundamentally change our strategies for the control of vector-borne diseases by facilitating rapid dissemination of transgenes that prevent pathogen transmission or reduce vector capacity. CRISPR/Cas9 gene drive promises such a mechanism, which works by converting cells that are heterozygous for the drive construct into homozygotes, thereby enabling super-Mendelian inheritance. Although CRISPR gene drive activity has already been demonstrated, a key obstacle for current systems is their propensity to generate resistance alleles, which cannot be converted to drive alleles. In this study, we developed two CRISPR gene drive constructs based on the nanos and vasa promoters that allowed us to illuminate the different mechanisms by which resistance alleles are formed in the model organism Drosophila melanogaster. We observed resistance allele formation at high rates both prior to fertilization in the germline and post-fertilization in the embryo due to maternally deposited Cas9. Assessment of drive activity in genetically diverse backgrounds further revealed substantial differences in conversion efficiency and resistance rates. Our results demonstrate that the evolution of resistance will likely impose a severe limitation to the effectiveness of current CRISPR gene drive approaches, especially when applied to diverse natural populations.

CRISPR homing gene drives can convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. Such a mechanism could be used, for example, to rapidly disseminate a genetic payload in a population, promising effective strategies for the control of vector-borne diseases. However, all CRISPR homing gene drives studied in insects thus far have produced significant quantities of resistance alleles that would limit their spread. In this study, we provide an experimental demonstration that multiplexing of guide RNAs can both significantly increase the drive conversion efficiency and reduce germline resistance rates of a CRISPR homing gene drive in We further show that an autosomal drive can achieve drive conversion in the male germline, with no subsequent formation of resistance alleles in embryos through paternal carryover of Cas9. Finally, we find that the promoter significantly lowers somatic Cas9 expression compared with the promoter, suggesting that provides a superior choice in drive strategies where gene disruption in somatic cells could have fitness costs. Comparison of drive parameters among the different constructs developed in this study and a previous study suggests that, while drive conversion and germline resistance rates are similar between different genomic targets, embryo resistance rates can vary significantly. Taken together, our results mark an important step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further control of resistance rates.

Gene drives could allow for control of vector-borne diseases by directly suppressing vector populations or spreading genetic payloads designed to reduce pathogen transmission. Clustered regularly interspaced short palindromic repeat (CRISPR) homing gene drives work by cleaving wild-type alleles, which are then converted to drive alleles by homology-directed repair, increasing the frequency of the drive in a population over time. However, resistance alleles can form when end-joining repair takes place in lieu of homology-directed repair. Such alleles cannot be converted to drive alleles, which would eventually halt the spread of a drive through a population. To investigate the effects of natural genetic variation on resistance formation, we developed a CRISPR homing gene drive in Drosophila melanogaster and crossed it into the genetically diverse Drosophila Genetic Reference Panel (DGRP) lines, measuring several performance parameters. Most strikingly, resistance allele formation postfertilization in the early embryo ranged from 7 to 79% among lines and averaged 42 6 18%. We performed a genome-wide association study using our results in the DGRP lines, and found that the resistance and conversion rates were not explained by common alleles of large effect, but instead there were several genetic polymorphisms showing weak association. RNA interference knockdown of several genes containing these polymorphisms confirmed their effect, but the small effect sizes imply that their manipulation would likely yield only modest improvements to the efficacy of gene drives.

Estimating fitness differences between allelic variants is a central goal of experimental evolution. Current methods for inferring such differences from allele frequency time series typically assume that the effects of selection can be described by a fixed selection coefficient. However, fitness is an aggregate of several components including mating success, fecundity, and viability. Distinguishing between these components could be critical in many scenarios. Here, we develop a flexible maximum likelihood framework that can disentangle different components of fitness from genotype frequency data, and estimate them individually in males and females. As a proof-of-principle, we apply our method to experimentally evolved cage populations of Drosophila melanogaster, in which we tracked the relative frequencies of a loss-of-function and wild-type allele of yellow. This X-linked gene produces a recessive yellow phenotype when disrupted and is involved in male courtship ability. We find that the fitness costs of the yellow phenotype take the form of substantially reduced mating preference of wild-type females for yellow males, together with a modest reduction in the viability of yellow males and females. Our framework should be generally applicable to situations where it is important to quantify fitness components of specific genetic variants, including quantitative characterization of the population dynamics of CRISPR gene drives.

CRISPR gene drives can efficiently convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. This mechanism could be used, for example, to rapidly disseminate a genetic payload through a population, promising novel strategies for the control of vector-borne diseases. However, all CRISPR gene drives tested have produced significant quantities of resistance alleles that cannot be converted to drive alleles and would likely prevent these drives from spreading in a natural population. In this study, we assessed three strategies for reducing resistance allele formation. First, we directly compared drives with the nanos and vasa promoters, which showed that the vasa drive produced high levels of resistance alleles in somatic cells. This was not observed in the nanos drive. Another strategy was the addition of a second gRNA to the drive, which both significantly increased the drive conversion efficiency and reduced the formation rate of resistance alleles. Finally, to minimize maternal carryover of Cas9, we assessed the performance of an autosomal drive acting in the male germline, and found no subsequent formation of resistance alleles in embryos. Our results mark a step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further reduction of resistance rates.