Abstract
Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.
Highlights
Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission
A mixture comprising a donor plasmid marked with green fluorescent protein (GFP) and two plasmids, each encoding one of the two gRNAs, was injected into 504 embryos of the DsRed-marked nRec gene-drive line, which carries vasa-Cas[9] and U6Akh2-gRNA transgenes targeting the kh locus in the germline[8]
As hypothesized, the rate of decrease exceeded that expected for the elimination of homozygous recessive individuals solely by negative selection and Mendelian inheritance. We propose that this is driven by lethal/ sterile mosaicism (Fig. 4a–c), which accelerates the elimination of kh− mutations inherited through females
Summary
Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. One population modification approach is to express multiple anti-parasite effector molecules in the form of single-chain antibodies directed against sexual stages of Plasmodium falciparum in relevant mosquito tissues[8,11,12] This strategy relies on efficient copying of a drive system from one chromosome to its homolog in the germline by homology-directed repair (HDR) following the induction of double-stranded DNA breaks by the Cas[9] endonuclease. The recoded kh sequence carried by the drive construct supports normal survival and reproductive capacity in females, while females failing to inherit the Reckh construct from their mothers and carrying nonfunctional mutated copies of kh are culled from the population This elimination does not rely solely on the standard negative selection of load-conferring recessive alleles but is driven by lethal/sterile mosaicism resulting from maternally deposited Cas9/gRNA complexes mutating the wild-type (WT) paternal allele. We report a Cas9/gRNAmediated cassette exchange[29] in mosquitoes called “Swap” that permits the rapid and flexible replacement of specific sequences within genomically integrated transgenes without the need for docking sites
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