A homing rescue gene drive with multiplexed gRNAs reaches high frequency in cage populations but generates functional resistance.

CRISPR homing gene drives have considerable potential for managing populations of medically and agriculturally significant insects. They operate by Cas9 cleavage followed by homology-directed repair, copying the drive allele to the wild-type chromosome and thus increasing in frequency and spreading throughout a population. However, resistance alleles formed by end-joining repair pose a significant obstacle. To address this, we create a homing drive targeting the essential hairy gene in Drosophila melanogaster. Nonfunctional resistance alleles are recessive lethal, while drive carriers have a recoded "rescue" version of hairy. The drive inheritance rate is moderate, and multigenerational cage studies show drive spread to 96%-97% of the population. However, the drive does not reach 100% due to the formation of functional resistance alleles despite using four gRNAs. These alleles have a large deletion but likely utilize an alternate start codon. Thus, revised designs targeting more essential regions of a gene may be necessary to avoid such functional resistance. Replacement of the rescue element's native 3' UTR with a homolog from another species increases drive inheritance by 13%-24%. This was possibly because of reduced homology between the rescue element and surrounding genomic DNA, which could also be an important design consideration for rescue gene drives.