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

• Published in: Cell reports (Cambridge) Vol. 39; no. 8; p. 110843
• Main Authors: López Del Amo, Víctor, Juste, Sara Sanz, Gantz, Valentino M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.05.2022
• Subjects: CRISPR; Drosophila; gene drives; germline; homology-directed repair; nickase; CRISPR; germline; Drosophila; CP: Microbiology; CP: Molecular biology; gene drives; homology-directed repair; nickase
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2022.110843

CRISPR-based gene-drives have been proposed for managing insect populations, including disease-transmitting mosquitoes, due to their ability to bias their inheritance toward super-Mendelian rates (>50%). Current technologies use a Cas9 that introduces DNA double-strand breaks into the opposing wild-type allele to replace it with a copy of the gene-drive allele via DNA homology-directed repair. However, the use of different Cas9 versions is unexplored, and alternative approaches could increase the available toolkit for gene-drive designs. Here, we report a gene-drive that relies on Cas9 nickases that generate staggered paired nicks in DNA to propagate the engineered gene-drive cassette. We show that generating 5′ overhangs in the system yields efficient allelic conversion. The nickase gene-drive arrangement produces large, stereotyped deletions that are advantageous to eliminate viable animals carrying small mutations when targeting essential genes. Our nickase approach should expand the repertoire for gene-drive arrangements aimed at applications in mosquitoes and beyond. [Display omitted] •Nickase versions of Cas9 trigger DNA homology-directed (HDR) repair in vivo•Paired DNA nicks into DNA promote gene-drive’s super-Mendelian inheritance•Efficient gene-drive propagation occurs when 5′ overhangs are generated•Nickase genedrive-producing 3′ overhangs do not trigger HDR Gene-drives using wild-type Cas9 offer solutions to fight vector-borne diseases, yet alternative strategies are needed to increase the available toolkit. López Del Amo et al. describe a nickase-based gene-drive system that promotes super-Mendelian inheritance of an engineered cassette in the Drosophila germ line, providing alternative designs for CRISPR-based gene-drive.