title: Targeted genome editing in Aedes aegypti using TALENs.
abstract: The Culicine mosquito, Aedes aegypti, is both a major vector of arthropod-borne viruses (arboviruses) and a genetic model organism for arbovirus transmission. TALE nucleases (TALENs), a group of artificial enzymes capable of generating site-specific DNA lesions, consist of a non-specific FokI endonuclease cleavage domain fused to an engineered DNA binding domain specific to a target site. While TALENs have become an important tool for targeted gene disruption in a variety of organisms, application to the mosquito genome is a new approach. We recently described the use of TALENs to perform heritable genetic disruptions in A. aegypti. Here, we provide detailed methods that will allow other research laboratories to capitalize on the potential of this technology for understanding mosquito gene function. We describe target site selection, transient embryo-based assays to rapidly assess TALEN activity, embryonic microinjection and downstream screening steps to identify target site mutations.
authors.affiliation: Fralin Life Science Institute, Dept. of Entomology, Virginia Tech, Blacksburg, VA 20461, United States.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Aryan
authors.affiliation: Fralin Life Science Institute, Dept. of Entomology, Virginia Tech, Blacksburg, VA 20461, United States.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Myles
authors.affiliation: Fralin Life Science Institute, Dept. of Entomology, Virginia Tech, Blacksburg, VA 20461, United States. Electronic address: zachadel@vt.edu.
authors.forename: Zach N
authors.initials: ZN
authors.lastname: Adelman
mesh_terms: D000330:Aedes / Q000196:embryology / Q000235:genetics*; D000818:Animals; D003851:Deoxyribonucleases / Q000737:chemistry; D004625:Embryo, Nonmammalian; D005260:Female; D008297:Male; D008845:Microinjections / Q000295:instrumentation; D016297:Mutagenesis, Site-Directed / Q000379:methods*
chemical_list: D003851:Deoxyribonucleases
keywords: Aedes aegypti; Gene editing; Meganuclease; Mosquito; TALE effector; TALEN