Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi
Contributed by Anthony A. James, October 26, 2015 (sent for review October 11, 2015; reviewed by Malcolm Fraser and Marcelo Jacobs-Lorena)
Science Sessions podcast
June 11, 2019
Significance
Malaria continues to impose enormous health and economic burdens on the developing world. Novel technologies proposed to reduce the impact of the disease include the introgression of parasite-resistance genes into mosquito populations, thereby modifying the ability of the vector to transmit the pathogens. Such genes have been developed for the human malaria parasite Plasmodium falciparum. Here we provide evidence for a highly efficient gene-drive system that can spread these antimalarial genes into a target vector population. This system exploits the nuclease activity and target-site specificity of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, which, when restricted to the germ line, copies a genetic element from one chromosome to its homolog with ≥98% efficiency while maintaining the transcriptional activity of the genes being introgressed.
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.
Acknowledgments
The authors are grateful to Judy Coleman and Thai Binh Pham for mosquito husbandry. Research was supported by grants from the NIH (AI070654 and NS029870), a generous gift from Drs. Sarah Sandell and Michael Marshall (to E.B.), the W. M. Keck Foundation, and the NIH National Institute of Allergy and Infectious Diseases (AI29746 and AI116433 to A.A.J.).
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Freely available online through the PNAS open access option.
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Published online: November 23, 2015
Published in issue: December 8, 2015
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Acknowledgments
The authors are grateful to Judy Coleman and Thai Binh Pham for mosquito husbandry. Research was supported by grants from the NIH (AI070654 and NS029870), a generous gift from Drs. Sarah Sandell and Michael Marshall (to E.B.), the W. M. Keck Foundation, and the NIH National Institute of Allergy and Infectious Diseases (AI29746 and AI116433 to A.A.J.).
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Competing Interests
Conflict of interest statement: E.B. and V.G. are authors of a patent applied for by the University of California, San Diego that relates to the mutagenic chain reaction.
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Cite this article
Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi
, Proc. Natl. Acad. Sci. U.S.A.
112 (49) E6736-E6743,
https://doi.org/10.1073/pnas.1521077112
(2015).
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