This application is in response to Program Announcement PA-03-127 (Integrative and Collaborative Approaches to Research) and has the long-term goal of developing algorithms for predicting the binding specificities of Cys2His2 zinc finger proteins, the most common DMA-binding domain encoded in the human genome. To do this, we propose to engineer and characterize the DMA-binding specificities of a large series of artificial Cys2His2 zinc finger proteins and to use this comprehensive dataset to develop and validate probabilistic DMA-recognition codes for Cys2His2 zinc fingers with greatly improved predictive power. The development of a large archive of engineered Cys2His2 zinc finger domains with well-defined specificities should also be generally useful to a large number of researchers interested in engineering customized """"""""designer"""""""" zinc finger nucleases (recently shown to be powerful reagents for inducing site- specific gene targeting in human cells). Working together with Consortium Members Dr. Gary Stormo (of the Washington University School of Medicine) and Dr. Scot Wolfe (of the University of Massachusetts Medical School), we will develop methodologies, reagents, data, and algorithms relevant to understanding the DMA-binding specificities of Cys2His2 zinc fingers. These activities will enhance the long-term goals of R01-funded research programs in all three laboratories. Specifically, the proposed project will provide important zinc finger reagents and binding specificity data that will facilitate the long-term goals of R01GM069906 (Studies of NRSF/REST Zinc Finger-DMA Interactions;PI: Joung) to understand naturally occurring extended zinc finger-DMA interfaces and to design highly specific engineered zinc finger domains capable of recognizing unique addresses within mammalian genomes. Similarly, these reagents and specificity data will also facilitate the development of dimeric engineered zinc finger proteins that can bind to and regulate a single endogenous gene in eukaryotic cells (R01GM068110: Dimeric Cys2His2 Zinc Finger Proteins for Gene Targeting;PI: Wolfe). Finally, the development of algorithms to predict DMA-binding specificities of eukaryotic zinc finger transcription factors extends a fundamental goal of R01 HG000249 (DMA pattern identification and analysis;PI: Stormo) to develop computational methods to determine the specificity of DMA-binding transcription factors and identify their regulatory sites within genome sequences.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Resource-Related Research Projects (R24)
Project #
5R24GM078369-04
Application #
7772289
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Tompkins, Laurie
Project Start
2007-03-01
Project End
2011-02-28
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
4
Fiscal Year
2010
Total Cost
$370,830
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Gupta, Ankit; Christensen, Ryan G; Bell, Heather A et al. (2014) An improved predictive recognition model for Cys(2)-His(2) zinc finger proteins. Nucleic Acids Res 42:4800-12
Gupta, Ankit; Christensen, Ryan G; Rayla, Amy L et al. (2012) An optimized two-finger archive for ZFN-mediated gene targeting. Nat Methods 9:588-90
Christensen, Ryan G; Gupta, Ankit; Zuo, Zheng et al. (2011) A modified bacterial one-hybrid system yields improved quantitative models of transcription factor specificity. Nucleic Acids Res 39:e83
Curtin, Shaun J; Zhang, Feng; Sander, Jeffry D et al. (2011) Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases. Plant Physiol 156:466-73
Sander, Jeffry D; Dahlborg, Elizabeth J; Goodwin, Mathew J et al. (2011) Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat Methods 8:67-9
Kim, Jin-Soo; Lee, Hyung Joo; Carroll, Dana (2010) Genome editing with modularly assembled zinc-finger nucleases. Nat Methods 7:91; author reply 91-2
Sander, Jeffry D; Reyon, Deepak; Maeder, Morgan L et al. (2010) Predicting success of oligomerized pool engineering (OPEN) for zinc finger target site sequences. BMC Bioinformatics 11:543
Zhang, Feng; Maeder, Morgan L; Unger-Wallace, Erica et al. (2010) High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proc Natl Acad Sci U S A 107:12028-33
Maeder, Morgan L; Thibodeau-Beganny, Stacey; Sander, Jeffry D et al. (2009) Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays. Nat Protoc 4:1471-501
Sander, Jeffry D; Zaback, Peter; Joung, J Keith et al. (2009) An affinity-based scoring scheme for predicting DNA-binding activities of modularly assembled zinc-finger proteins. Nucleic Acids Res 37:506-15

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