Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis inAedes aegypti

• DOI: 10.1073/pnas.1502370112
• Publication Date: 2015-03-17T02:56:33Z
• Authors: Sanjay Basu; Azadeh Aryan; Justin M. Overcash; Glady Hazitha Samuel; Michelle A. E. An...; Timothy J. Dahlem; Kevin M. Myles; Zach N. Adelman
• Keywords: Recombination, Genetic; 0301 basic medicine; Genome; Base Sequence; DNA Repair; Genetic Vectors; Molecular Sequence Data; Temperature; Exons; Polymerase Chain Reaction; 3. Good health; Mutagenesis, Insertional; 03 medical and health sciences; Aedes; Mutation; Animals; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Silencing; RNA Editing; Transgenes; Dimerization; Plasmids; RNA, Double-Stranded

SignificanceMosquitoes are vectors of both parasites and viruses responsible for high-impact diseases including malaria, dengue, and chikungunya. Novel interventions based on genetic modification of the mosquito genome are currently being developed and implemented. To comprehensively exploit such interventions, detailed knowledge of mosquito physiology, genetics, and genome engineering are required. We developed and validated a two-step process for performing high-efficiency site-specific insertion of genetic material into the mosquito genome by first evaluating candidate site-specific nucleases in a rapid format, followed by germ line-based editing where the choice of DNA repair response is constrained. This model should significantly accelerate both basic and applied research concerning disease vector mosquitoes.