Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquitoAnopheles 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.

Genetics‐based methods for agricultural insect pest management

“…These gene‐editing approaches have been developed in mosquitoes either to suppress vector populations, by affecting female fertility (Burt, 2003; Deredec et al ., 2008; Hammond et al ., 2016), or to modify a population, by spreading a trait that affects the ability to harbour pathogens (Gantz et al ., 2015). Gene‐editing approaches could also be used to suppress agricultural pests and/or manage resistance; for example, CRISPR gene editing has been used in a functional study to identify suitable gene targets in diamondback moth (Huang et al ., 2016).…”

Behind New Zealand’s wild plan to purge all pests

“…Then, using what is known as a gene drive, scientists could engineer that gene to spread through the population. "It can go from 1% to 100% of the population in around 10 generations, " says Ethan Bier, a geneticist at the University of California, San Diego, who is using gene drives to engineer mosquitoes that are resistant to the malaria parasite 5 .…”

Bioethics for Biotechnologists: From Dolly to CRISPR

“…The aim is to spread deleterious traits or resistance genes for diseases among wild populations. The approach has been proved to be efficient; researchers at the University of California San Diego recently used the CRISPR/Cas9 system to engineer a gene drive to introduce two genes that confer resistance to malaria in the mosquito vector for the disease Anopheles gambiae (Gantz et al 2015). Although the aim of such research seeks to benefit humans, the consequences of eradicating species from the ecological web are mostly unknown, making risk prediction a difficult task.…”