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.

Public Health Relevance

Gene targeting by homologous recombination is a powerful tool that has been used extensively to create gene targeted mouse models of human disease. Aided by recent advances in engineered nuclease technology, this proposal seeks to develop broadly applicable methods that would enable the routine use of gene targeting in human somatic and pluripotent cells (and other cell types) to create relevant disease models, to functionally interrogate and study genetic variants in relation to human disease, and to genetically engineer safer cells for clinical therapeutic applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM105189-01
Application #
8456449
Study Section
Special Emphasis Panel (ZRG1-F08-Q (20))
Program Officer
Reddy, Michael K
Project Start
2013-02-01
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
1
Fiscal Year
2013
Total Cost
$52,190
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Tsai, Shengdar Q; Zheng, Zongli; Nguyen, Nhu T et al. (2015) GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33:187-97
Tsai, Shengdar Q; Wyvekens, Nicolas; Khayter, Cyd et al. (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol 32:569-76
Low, Benjamin E; Krebs, Mark P; Joung, J Keith et al. (2014) Correction of the Crb1rd8 allele and retinal phenotype in C57BL/6N mice via TALEN-mediated homology-directed repair. Invest Ophthalmol Vis Sci 55:387-95
Maeder, Morgan L; Angstman, James F; Richardson, Marcy E et al. (2013) Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat Biotechnol 31:1137-42