View Program - 63rd Annual Drosophila Research Conference

CRISPR gene drive systems can potentially modify an entire population due to their self-propagation capacity, representing a powerful tool to combat public health issues such as mosquito-borne diseases. Gene drive elements bias Mendelian inheritance (50%) towards super-Mendelian rates (~100%), allowing them to spread through populations. Traditional gene drives can replace wildtype alleles by performing DNA double-strand breaks and subsequent homology-directed repair (HDR). When the HDR is not precise, mutations at the target site can generate resistant alleles, preventing a gene drive's efficient spread.

We hypothesize that simultaneous paired-nicks targeting two adjacent DNA regions should generate a staggering double-strand break, and the subsequent DNA repair by HDR will promote the gene-drive super-Mendelian inheritance. Here, we developed a proof-of-concept nickase-based gene drive strategy that enables super-Mendelian inheritance of an engineered allele using Drosophila melanogaster. Additionally, we analyzed the quality of potential resistant alleles formed by our nickase gene-drive system and compared them to the traditional gene drive to explore potential advantages. In fact, we demonstrate for the first time that both nD10A and nH840A promote HDR in the germline using CRISPR gene drives, yielding comparable super-Mendelian inheritance rates to traditional gene drives. Furthermore, the double-nicking nature of this arrangement produced large deletions when using the nickase H840A combined with paired-gRNAs in a PAM-in configuration. This outcome provides potential advantages to target essential genes.

Indeed, our nickase gene-drive design should expand our alternatives for gene drive applications and other germline editing purposes in a broad range of organisms.

A nickase Cas9 gene-drive system promotes super-Mendelian inheritance in Drosophila melanogaster.