title: Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.
abstract: Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.
authors.affiliation: Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093-0349;
authors.forename: Valentino M
authors.initials: VM
authors.lastname: Gantz
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900;
authors.forename: Nijole
authors.identifier: http://orcid.org/0000-0002-0862-0349
authors.initials: N
authors.lastname: Jasinskiene
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900;
authors.forename: Olga
authors.initials: O
authors.lastname: Tatarenkova
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900;
authors.forename: Aniko
authors.initials: A
authors.lastname: Fazekas
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900;
authors.forename: Vanessa M
authors.initials: VM
authors.lastname: Macias
authors.affiliation: Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093-0349; ebier@ucsd.edu aajames@uci.edu.
authors.forename: Ethan
authors.initials: E
authors.lastname: Bier
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900; Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697-4500 ebier@ucsd.edu aajames@uci.edu.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: James
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000852:Anopheles / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000502:physiology*; D005260:Female; D007303:Insect Vectors; D008288:Malaria / Q000517:prevention & control* / Q000635:transmission; D008297:Male; D009032:Mosquito Control / Q000379:methods*
keywords: CRISPR; MCR; Plasmodium falciparum; eradication; transgenesis