CRISPR-SD, a programmable Cas9 approach to generating X-shredder lines in insects with XY mating systems

Bill Reid, Ph.D. Postdoctoral Researcher, Institute for Bioscience and Biotechnology Research, University of Maryland College Park

Bill Reid, Ph.D. Postdoctoral Researcher, Mosquito and Fly Research Unit, USDA/ARS, Center for Agricultural, Medical and Veterinary Entomology, Gainesville, Florida MORE ABOUT THE AUTHOR

In a recent paper in Scientific Reports, Galizi, Hammond, et al report on the use of CRISPR-Cas9 to develop a sex-distortion system in Anopheles gambiae, which they termed CRISPRSD. Earlier, the group had demonstrated that transgenic An. gambaie expressing the endonuclease I-PpoI could be used to generate “X-shredder” lines of An. gambiae. In this system, I-Ppol, which targets critical sequences on the X chromosome in An. gambiae, was expressed under control of a promoter that restricted the expression of the endonuclease to male meiosis, resulting in a favoring of Y-bearing spermatozoa, resulting in predominantly male offspring.

Galizi_blog_Fig1

Targeting of the X chromosome using male meiotic expressed I-Ppol in An. gambiae resulting in Y-bearing spermatozoa only. Image from Galizi et al (2014).

By expressing Cas9 using the same male meiotic promoter, and sgRNA guides under control of a ubiquitously-expressed U6 PolIII promoter, the researchers were able to replicate the same phenotype, establishing four lines of An. gambiae that exhibited male biases ranging from 86.1-94.8%, with three of these lines showing good male fertility, and a fourth with reduced fertility, which the authors attributed to possible position effects.

A selfish gene (blue) spreading through a mosquito population

A selfish gene (blue) spreading through a mosquito population

While the authors point out several current limitations of the system for pest management (development of cleavage-resistant alleles, full inheritance only if Y-linked, etc.), the demonstration of the utility of using CRISPR-Cas9 to generate sex-ratio distortions in insects with XY mating systems removes the limitation of the requirement that unique endonuclease targets must first be present on the X chromosome. Through careful bioinformatic analyses and validation testing in the whole animal, the direct targeting of X-linked essential genes, such as the 28S rDNA (which was the target for this work), represents an interesting approach to consider if interested in generating sex-distorted lines of insects with XY mating systems.

Galizi R, Hammond A, Kyrou K, Taxiarchi C, Bernardini F, O’Loughlin SM, Papathanos P-A, Nolan T, Windbichler N, Crisanti A. 2016. A CRISPR-Cas9 sex-ratio distortion system for genetic control. Scientific Reports 6:31139. DOI: 10.1038/srep31139.

Galizi R, Doyle LA, Menichelli M, Bernardini F, Deredec A, Burt A, Stoddard BL, Windbichler N, Crisanti A. 2014. A synthetic sex ratio distortion system for the control of the human malaria mosquito.Nature Communications 5, Article number: 3977. doi:10.1038/ncomms4977

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