PT  - JOURNAL ARTICLE
AU  - Kathryn E. Kistler
AU  - Leslie B. Vosshall
AU  - Benjamin J. Matthews
TI  - Genome-engineering with CRISPR-Cas9 in the mosquito <em>Aedes aegypti</em>
AID  - 10.1101/013276
DP  - 2014 Jan 01
TA  - bioRxiv
PG  - 013276
4099  - http://biorxiv.org/content/early/2014/12/27/013276.short
4100  - http://biorxiv.org/content/early/2014/12/27/013276.full
AB  - The mosquito Aedes aegypti is a potent vector of the Chikungunya, yellow fever, and Dengue viruses, which result in hundreds of millions of infections and over 50,000 human deaths per year. Loss-of-function mutagenesis in Ae. aegypti has been established with TALENs, ZFNs, and homing endonucleases, which require the engineering of DNA-binding protein domains to generate target specificity for a particular stretch of genomic DNA. Here, we describe the first use of the CRISPR-Cas9 system to generate targeted, site-specific mutations in Ae. aegypti. CRISPR-Cas9 relies on RNA-DNA base pairing to generate targeting specificity, resulting in cheaper, faster, and more flexible genome-editing reagents. We investigate the efficiency of reagent concentrations and compositions, demonstrate the ability of CRISPR-Cas9 to generate several different types of mutations via disparate repair mechanisms, and show that stable germ-line mutations can be readily generated at the vast majority of genomic locitested. This work offers a detailed exploration into the optimal use of CRISPR-Cas9 in Ae. aegypti that should be applicable to non-model organisms previously out of reach of genetic modification.