A synthetic homing endonuclease-based gene drive system in the human malaria mosquito

Abstract

Genetic methods of manipulating or eradicating disease vector populations have long been discussed as an attractive alternative to existing control measures because of their potential advantages in terms of effectiveness and species specificity1–3. The development of genetically engineered malaria-resistant mosquitoes has shown, as a proof-of-principle, the possibility of targeting the mosquito’s ability to serve as a disease vector4–7. The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations8. We have previously suggested that homing endonuclease genes (HEGs), a class of simple selfish genetic elements, could be exploited for this purpose9. Here we demonstrate that a synthetic genetic element, consisting of mosquito regulatory regions10 and the homing endonuclease gene I-SceI11–13, can substantially increase its transmission to the progeny in transgenic mosquitoes of the human malaria vector Anopheles gambiae. We show that the I-SceI element is able to rapidly invade receptive mosquito cage populations, validating mathematical models for the transmission dynamics of HEGs. Molecular analyses confirm that expression of I-SceI in the male germline induces high rates of site-specific chromosomal cleavage and gene conversion, which results in the gain of the I-SceI gene, and underlies the observed genetic drive. These findings demonstrate a new mechanism by which genetic control measures can be implemented. Our results also show in principle how sequence-specific genetic drive elements like HEGs could be used to take the step from the genetic engineering of individuals to the genetic engineering of populations.

Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations

“…No engineered endonuclease gene drive capable of spreading through a wild population has yet been published. However, the Crisanti and Russell laboratories have constructed gene drives that can only spread through laboratory mosquito 9 and fruit fly 16,63 populations that have been engineered to contain the endonuclease cut site. The Burt and Crisanti laboratories are attempting to build a male-biasing suppression drive using an endonuclease that serendipitously cuts a conserved sequence repeated hundreds of times in the X chromosome of the mosquito Anopheles gambiae 7,10,64 .…”

“…Incomplete cutting was a problem for the homing endonuclease drive constructed in transgenic mosquitoes (72% cutting) and also for the homing endonuclease, zinc-finger nuclease, and TALEN drives in fruit flies (37%, 86%, and 70% cutting) 9,16,76 . The simplest way to increase cutting is to target multiple adjacent sequences.…”

“…These genes bias inheritance by cutting the homologous chromosome, inducing the cell to copy them when it repairs the break. Several efforts have focused on the possibility of using gene drives targeting mosquitoes to block malaria transmission [7][8][9][10][11][12] . However, development has been hindered by the difficulty of engineering homing endonucleases to cut new target sequences [13][14][15] .…”

Genome-Editing Technologies: Principles and Applications

“…Finally, targeted nucleases have also been used to inactivate pathogenic genes to prevent viral (Lin et al 2014) or parasitic (Ghorbal et al 2014) infection, as well as to introduce knockin-specific factors capable of imparting pathogen resistance . Intriguingly, targeted nucleases could also serve as conduits for curbing mosquito-or insect-borne diseases through a technique known as gene drive (Burt 2003;Sinkins and Gould 2006), which harnesses genome editing to facilitate the introduction of a specific gene or mutation that can then confer a particular phenotype into a host and also be transmitted to its progeny (Windbichler et al 2011). Gene drives have now been tested in the malaria vector mosquitos Anopheles stephensi and Anopheles gambiae (Hammond et al 2016) as a means for achieving population control and to prevent disease transmission, respectively.…”

Efficient ΦC31 integrase–mediated site-specific germline transformation of Anopheles gambiae

“…In most studies, in vitro produced mRNA is used as a source of integrase 33,34 , but plasmid templates have recently also proved suitable (A. L., unpublished ; S. Fuchs, T. Nolan & A.…”