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

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

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Hou S, Chen J, Feng R, Xu X, Liang N, Champer J.,  Journal of Genetics and Genomics,  2024.

CRISPR homing gene drive is a potent technology with considerable potential for managing populations of medically and agriculturally significant insects. It induces a bias in the inheritance of the drive allele in progeny, rapidly spreading the desired gene construct throughout the population. Homing drives operate by Cas9 cleavage followed by homology-directed repair, copying the drive allele to the wild-type chromosome. However, resistance alleles formed by end-joining repair pose a significant obstacle to the spread of the drive. To address this challenge, we create a homing drive targeting the essential but haplosufficient hairy gene in Drosophila melanogaster. Our strategy involves eliminating nonfunctional resistance alleles, which are recessive lethal, while rescuing drive-carrying individuals with a recoded 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 fails to reach the whole population due to the formation of functional resistance alleles, despite use of four gRNAs. These alleles have a large deletion but likely utilize an alternate start codon, and they have a small fitness advantage over the drive.

Thus, revised design strategies targeting more essential regions of a target gene may often be necessary to avoid such functional resistance, even when using multiplexed gRNAs. Replacement of the native 3′ UTR of the rescue element with a homolog from another species increases drive inheritance by 13%-24%. This was possibly because of reduced homology between the rescue element and nearby native DNA, which could also be an important design consideration for gene drives that utilize rescue elements.