Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with their abilities to replicate indefinitely and maintain pluripotency, offer tremendous opportunities for the development of novel regenerative medicine-based therapies. iPSC technology, in particular, may provide the means to develop personalized therapeutic strategies in which a patient's own cells might be modified and used to treat their disease without the need for immunosuppression. Patient-derived ESCs and iPSCs will also provide critically important reagents for modeling and studying the biology of various diseases. Despite tremendous recent progress, many significant challenges remain to be addressed before ESCs and iPSCs can be used routinely for therapeutics. For example, methods for generating iPSCs are currently inefficient with considerable room for improvement. In addition, homologous recombination-mediated gene targeting for creating specific mutations works with low efficiency and is difficult to perform, particularly in human ESCs and iPSCs. Lastly, methods for robustly differentiating ESCs and iPSCs into cell types of interest are typically inefficient and yield cells that fail to exhibit all desired characteristics. The goal of this proposal is to employ engineered zinc finger technology to address these major challenges impeding research and therapeutic applications of ESCs and iPSCs. Specifically, this proposal will utilize the PI's expertise in engineered zinc finger technology to address three important challenges for stem cell biology and research: (1) developing more efficient methods for iPSC generation, (2) enabling high efficiency ZFN-induced gene targeting in human ESCs and iPSCs, and (3) creating more efficient methods for differentiating stem cells into desired cell types of interest. Public Health Relevance: This application seeks to address significant challenges that currently limit research and therapeutic applications of human embryonic stem cells a

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
NIH Director’s Pioneer Award (NDPA) (DP1)
Project #
Application #
Study Section
Special Emphasis Panel (ZGM1-NDPA-B (01))
Program Officer
Haynes, Susan R
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Massachusetts General Hospital
United States
Zip Code
Kleinstiver, Benjamin P; Pattanayak, Vikram; Prew, Michelle S et al. (2016) High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490-5
Kleinstiver, Benjamin P; Tsai, Shengdar Q; Prew, Michelle S et al. (2016) Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34:869-74
Zuris, John A; Thompson, David B; Shu, Yilai et al. (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33:73-80
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
Gjini, Evisa; Mansour, Marc R; Sander, Jeffry D et al. (2015) A zebrafish model of myelodysplastic syndrome produced through tet2 genomic editing. Mol Cell Biol 35:789-804
Keung, Albert J; Joung, J Keith; Khalil, Ahmad S et al. (2015) Chromatin regulation at the frontier of synthetic biology. Nat Rev Genet 16:159-71
Wyvekens, Nicolas; Tsai, Shengdar Q; Joung, J Keith (2015) Genome Editing in Human Cells Using CRISPR/Cas Nucleases. Curr Protoc Mol Biol 112:31.3.1-18
Liao, Jing; Karnik, Rahul; Gu, Hongcang et al. (2015) Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells. Nat Genet 47:469-78
Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q et al. (2015) Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523:481-5
Spisák, Sándor; Lawrenson, Kate; Fu, Yanfang et al. (2015) CAUSEL: an epigenome- and genome-editing pipeline for establishing function of noncoding GWAS variants. Nat Med 21:1357-63

Showing the most recent 10 out of 42 publications