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

Abstract

CRISPR-based gene drive systems can be used to modify entire wild populations due to their ability to bias their own inheritance towards super-Mendelian rates (>100%). Current gene drives contain a Cas9 and a gRNA gene inserted at the location targeted by the gRNA. These gene products are able to cut the opposing wildtype allele, and lead to its replacement with a copy of the gene drive through the homology-directed DNA repair pathway. When this allelic conversion occurs in the germline it leads to the preferential inheritance of the engineered allele — a property that has been proposed to disseminate engineered traits for managing disease-transmitting mosquito populations. Here, we report a novel gene-drive strategy relying on Cas9 nickases which operates by generating staggered paired-nicks in the DNA to promote propagation of the gene drive allele. We show that only when 5’ overhangs are generated, the system efficiently leads to allelic conversion. Further, the nickase gene-drive arrangement produces large stereotyped deletions, providing potential advantages for targeting essential genes. Indeed, the nickase-gene-drive design should expand the options available for gene drive designs aimed at applications in mosquitoes and beyond.

Cas9/Nickase-induced allelic conversion by homologous chromosome-templated repair in Drosophila somatic cells

“…We took our analysis further by comparing the relative efficiencies of HTR processes following DSB or nicks targeting opposing DNA strands. For these experiments, we made use of three equivalent transgenic cassettes inserted at the same site to express either Cas9, D10A, or the alternate H840A nickase at identical levels to repair the CS1 − allele ( 23 ). H840A is mutated in the HNH catalytic domain and thus produces SSB on the opposite nontargeted DNA strand from that cleaved by D10A.…”

“…For comparing the activity of the different nucleases and deep sequencing analysis (Fig. 3), three lines were designed in which vasaCas9, vasaD10A, or vasaH840A sequences associated with the DsRed marker were inserted at the same location in the yellow gene ( 23 ). For pairing-independent HDR (Fig.…”

The Challenges in Developing Efficient and Robust Synthetic Homing Endonuclease Gene Drives

“…These results indicate that any individual cut site in a classical multiplexing may be less efficient than a drive element optimised for only one cut site. Nonetheless, additional studies have indicated that multiplexing can increase the overall efficiency of a HEG albeit in some cases with diminishing returns for additional gRNAs ( Champer et al, 2018 , Champer et al, 2020 S. E. ; Yang et al, 2021 ), and ( López Del Amo et al, 2021 ) compared to ( López Del Amo et al, 2020b ).…”