Journal Article

Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti

Bowman, Christian S., Akbari, Omar S., Li, Ming, White, Bradley J., Yang, Ting, Bui, Michelle

2017

Overview

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti. Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.

• Publisher: National Academy of Sciences
• Subject: Aedes - genetics / Aedes - growth & development / Aedes - metabolism / Aedes aegypti / Animals / Aquatic insects / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Biological Sciences / Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR / CRISPR-Associated Protein 9 / CRISPR-Cas Systems / Efficiency / Embryos / Endonucleases - genetics / Endonucleases - metabolism / Female / Founder Effect / Gene Editing - methods / Gene expression / Gene Expression Regulation / Genes / Genetic screening / Genetics / Genome, Insect / Genomes / Germ-Line Mutation / Homology / Injection / Male / Morphology / Mosquito Vectors - genetics / Mosquito Vectors - growth & development / Mosquito Vectors - metabolism / Mosquitoes / PNAS Plus / Population control / Promoter Regions, Genetic / Recombinational DNA Repair / Reverse Genetics - methods / RNA, Guide, CRISPR-Cas Systems - genetics / RNA, Guide, CRISPR-Cas Systems - metabolism / Strains (organisms)