The long-term objective of this proposal is to develop broadly applicable methods to utilize engineered nucleases to facilitate gene targeting in human somatic and pluripotent cells. The specific aims are: 1) to enhance our ability to genetically engineer all types of genetic modifications by homologous recombination using engineered nucleases (TAL effector nucleases or TALENs) in human somatic and pluripotent cells and 2) to improve our ability to use TALEN-derived nicking enzymes for homologous recombination. This proposal will leverage a high-throughput assembly method for creating engineered TALENs and apply it to the genetic engineering of human cell lines. Successful development of the proposed methods for introducing small insertions and single-base modifications to the human genome will enhance the ability to systematically investigate the relationship between human gene variation and function and enable genetic manipulations that have been previously limited to mouse model systems.