Rapid assembly of customized TALENs into multiple delivery systems.
Transcriptional activator-like effector nucleases (TALENs) have become a powerful tool for genome editing. Here we present an efficient TALEN assembly approach in which TALENs are assembled by direct Golden Gate ligation into Gateway(®) Entry vectors from a repeat variable di-residue (RVD) plasmid array. We constructed TALEN pairs targeted to mouse Ddx3 subfamily genes, and demonstrated that our modified TALEN assembly approach efficiently generates accurate TALEN moieties that effectively introduce mutations into target genes. We generated "user friendly" TALEN Entry vectors containing TALEN expression cassettes with fluorescent reporter genes that can be efficiently transferred via Gateway (LR) recombination into different delivery systems. We demonstrated that the TALEN Entry vectors can be easily transferred to an adenoviral delivery system to expand application to cells that are difficult to transfect. Since TALENs work in pairs, we also generated a TALEN Entry vector set that combines a TALEN pair into one PiggyBac transposon-based destination vector. The approach described here can also be modified for construction of TALE transcriptional activators, repressors or other functional domains.
Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute and Foundation, Pittsburgh, Pennsylvania, United States of America.
Zhengxing
Z
Zhang
Siliang
S
Zhang
Xin
X
Huang
Kyle E
KE
Orwig
Yi
Y
Sheng
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D016503:Drug Delivery Systems / Q000379:methods; D004720:Endonucleases / Q000008:administration & dosage* / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D051379:Mice
D004720:Endonucleases