CRISPR-based homing gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population. Here, we experimentally demonstrate the suitability of synthetic target site drives as well as split drives as flexible safeguarding strategies for gene drive experiments by showing that their performance closely resembles that of standard homing drives in Drosophila melanogaster. Using our split drive system, we further find that maternal deposition of both Cas9 and gRNA is required to form resistance alleles in the early embryo and that maternally-deposited Cas9 alone can power germline drive conversion in individuals that lack a genomic source of Cas9.
CRISPR-based gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population.Here, we experimentally demonstrate the suitability of synthetic target sites and split drives as flexible safeguarding strategies for gene drive experiments.
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.
Engineered gene drives have been suggested as a mechanism for rapidly spreading genetic alterations through a population. One promising type of drive is the CRISPR homing drive, which has recently been demonstrated in several organisms. However, such drives face a major obstacle in the form of resistance against the drive that typically evolves rapidly. In addition, homing-type drives are generally self-sustaining, meaning that a drive would likely spread to all individuals of a species even when introduced at low frequency in a single location. Here, we develop a new form of CRISPR gene drive, the Toxin-Antidote Recessive Embryo (TARE) drive, which successfully limits resistance by targeting a recessive lethal gene while providing a recoded sequence to rescue only drive-carrying individuals. Our computational modeling shows that such a drive will have threshold-dependent dynamics, spreading only when introduced above a frequency threshold that depends on the fitness cost of the drive. We demonstrate such a drive in Drosophila with 88-95% transmission to the progeny of female drive heterozygotes. This drive was able to spread through a large cage population in just six generations following introduction at 24% frequency without any apparent evolution of resistance. Our results suggest that TARE drives constitute promising candidates for the development of effective, regionally confined population modification drives.One possible strategy for reducing resistance potential is to remove the need for homologydirected repair altogether. This criterion is fulfilled by drives using the "toxin-antidote" principle.
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