Cas9-Mediated Gene-Drive In Mosquitoes

Frank Crisione

Frank Crisione, Ph.D., Postdoctoral Research Associate, Institute for Bioscience and Biotechnology Research, University of Maryland College Park. More about the Author

Gantz and Bier who demonstrated the fundamental principles of a Cas9-based gene drive system using Drosophila melanogaster in a publication earlier this year have recreated that system in the mosquito Anopheles stephensi with the help of collaborators.  Gantz et al. (2015)

The potential to control malaria using transgenic insect technologies is often raised in the vector biology community but posses many technical issues. One issue is cost-effective sustainability. Should a mosquitoes incapable of transmitting malaria be created, the question of how the genotype would be propagated and sustained in natural population has fostered interest in gene-drives, genetic systems that skew transmission ratios in favor of some alleles over others. Basically, gene-drives are ‘gene-spreading systems’.  Gene-drive systems linked to ‘phenotype effectors’ require few releases of transgenic mosquitoes in malaria endemic areas, increasing the systems sustainability.

A Homing Endonuclease increasing in frequency with a genome.

A Homing Endonuclease increasing in frequency with a genome.

Homing endonucleases are well-known and well-studied gene drive systems and programmable endonucleases such as Cas9 can be used to recreate such systems with user-defined homing characteristics. This is what Gantz et al have done in Anopheles stephensi.

A selfish gene (blue) spreading through a mosquito population

A selfish gene (blue) spreading through a mosquito population

The system’s “drive” is achieved by the expression of Cas9 protein in conjunction with specific guide RNAs and flanked by homology arms to the desired site of integration. The cassette is designed so that it can “autonomously” integrate itself into the target region of the genome and cheat classical Mendelian genetics by converting its partner wild-type allele into another drive cassette through homology driven repair mechanisms.

The system described by Gantz et al. targets the kynurenine hydrolase gene resulting in an easily recognized eye-color phenotype when both alleles are mutated by the drive system (a convenient visual monitoring system for spread in laboratory populations).  In the authors original publication the eye pigmentation gene white in Drosophila melanogaster was targeted for the same reasons.

Gene Drive

Synthetic gene drive system based on site specific DNA endonucleases (homing endonucleases, ZFN, TALENS, Cas9). Target sites and homology arms are chosen so that the endonuclease and associated genes (gRNA and other transgenes) are copied into chromosomes at the target site. Image credit – Dr. William Reid

Gantz et al  have also included a pair of genes coding for single chain antibodies that have been shown to antogonize Plasmodium infections in mosquitoes however the ability of the mosquitoes created for this study to support and transmit Plasmodium are not reported.

Besides demonstrating the feasibility of rapidly assembling a Cas9-based gene drive system in an important insect species there are valuable data and resources for scientists working with CRISPR-Cas9 and mosquitoes.  In order to construct their system Gantz et al. identified and isolated the Anopheles stephensi U6A promoter – a PolIII promoter useful for expressing gRNA in vivo.  The investigators use the promoter from the A. stephensi gene vasa to regulate expression of Cas9 and saw some maternal effects that were interesting and important.  Finally, they report the efficiency of homology dependent repair (drive efficiency) over the course of 3 generations in populations on the order of 1000 individuals and found it to be quiet high – ~98%.

The significance of this publication is in demonstrating the flexibility and ease of assembly of a Cas9 gene-drive system.

Gene-drives as a ‘pest’ control tool has become topic of controversy now that these systems can be readily assembled and deployed in some species.  As with the release of many transgenic organisms into the environment, they cannot be recalled. Unlike the average transgenic organism, organisms carrying gene drives spread at a much higher rate than their wild-type counterparts and saturate a population in much fewer generations.

How and when to use this technology is much-discussed and Gantz et al’s publication will keep that conversation going.

Valentino M. Gantz, Nijole Jasinskiene, Olga Tatarenkova, Aniko Fazekas, Vanessa M. Macias, Ethan Bier, and Anthony A. James (2015) Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi PNAS 2015 ; published ahead of print November 23, 2015, doi:10.1073/pnas.1521077112

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