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

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.


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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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