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 homing gene drives potentially have the capacity for large-scale population modification or suppression. However, resistance alleles formed by the drives can prevent them from successfully spreading. Such alleles have been found to form at high rates in most studies, including those in both insects and mammals. One possible solution to this issue is the use of multiple guide RNAs (gRNAs), thus allowing cleavage by the drive even if resistance sequences are present at some of the gRNA target sequences. Here, we develop a high-fidelity model incorporating several factors affecting the performance of drives with multiple gRNAs, including timing of cleavage, reduction in homology-directed repair efficiency due to imperfect homology around the cleavage site, Cas9 activity saturation, variance in the activity level of individual gRNAs, and formation of resistance alleles due to incomplete homology-directed repair. We parameterize the model using data from homing drive experiments designed to investigate these factors and then use it to analyze several types of homing gene drives. We find that each type of drive has an optimal number of gRNAs, usually between two and eight, dependent on drive type and performance parameters. Our model indicates that utilization of multiple gRNAs is insufficient for construction of successful gene drives, but that it provides a critical boost to drive efficiency when combined with other strategies for population modification or suppression.

The rapid evolution of resistance alleles poses a major obstacle for genetic manipulation of populations with CRISPR homing gene drives. One proposed solution is using multiple guide RNAs (gRNAs), allowing a drive to function even if some resistant target sites are present. Here, we develop a model of homing mechanisms parameterized by experimental studies. Our model incorporates several factors affecting drives with multiple gRNAs, including timing of cleavage, reduction in homology-directed repair efficiency due to imperfect homology, Cas9 activity saturation, gRNA activity level variance, and incomplete homology-directed repair. We find that homing drives have an optimal number of gRNAs, usually between two and eight, depending on the specific drive type and performance parameters. These results contradict the notion that resistance rates can be reduced to arbitrarily low levels by gRNA multiplexing and highlight the need for combined approaches to counter resistance evolution in CRISPR homing drives.

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.