Novel DNA-binding specificity can be engineered into Cys2His2 zinc finger proteins by either selection or design, and they can be used to deliver a DNA cleavage domain to a specific "address" in a genome. These tailor-made restriction endonucleases ("Zinc Finger Nucleases" or ZFNs), which function as (hetero)dimers, can introduce double stranded breaks at a specific genomic location to inactivate a target gene through imprecise repair or recode a target gene through homologous recombination with an exogenously supplied donor DNA. ZFN technology can potentially be used to apply simple or sophisticated reverse genetic approaches to a broad range of metazoan systems that were previously only accessible in the mouse and fly. This technology, which should also find application in bioengineering and human gene therapy, is still in its infancy. The current generation of ZFNs has not been thoroughly characterized nor completely optimized. We propose to develop a new generation of artificial nucleases with improved properties (activity, precision, range of targetable sequences) that will fully realize the potential of this technology for tailored genome editing.
In Aims 1 &2 we will define the optimal assembly of ZFPs and the optimal fusion of the nuclease domain to create heterodimeric ZFNs that are both efficient and precise. We will also develop new nuclease architectures to expand the types of DNA sequences that can be targeted.
In Aim 3 we will explore the potential of ZFNs to perform knockouts of highly related gene families, which would allow complex knockout combinations to be rapidly created.

Public Health Relevance

This research focuses on the development of artificial proteins that can be targeted to a unique site in a vertebrate genome to alter its content. This technology has potential application in human gene therapy, but much work remains to define versions of these proteins that will be highly active, yet will not damage other parts of the genome. Understanding how to improve the characteristics of these artificial systems will be the focus of this study.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068110-08
Application #
8197657
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Reddy, Michael K
Project Start
2004-02-01
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
8
Fiscal Year
2012
Total Cost
$325,710
Indirect Cost
$127,710
Name
University of Massachusetts Medical School Worcester
Department
Genetics
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
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
Kearns, Nicola A; Genga, Ryan M J; Enuameh, Metewo S et al. (2014) Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. Development 141:219-23
Zhu, Cong; Gupta, Ankit; Hall, Victoria L et al. (2013) Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases. Nucleic Acids Res 41:2455-65
Enuameh, Metewo Selase; Asriyan, Yuna; Richards, Adam et al. (2013) Global analysis of Drosophila Cys?-His? zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants. Genome Res 23:928-40
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
Gupta, Ankit; Meng, Xiangdong; Zhu, Lihua J et al. (2011) Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases. Nucleic Acids Res 39:381-92
Noyes, Marcus B; Meng, Xiangdong; Wakabayashi, Atsuya et al. (2008) A systematic characterization of factors that regulate Drosophila segmentation via a bacterial one-hybrid system. Nucleic Acids Res 36:2547-60
Meng, Xiangdong; Noyes, Marcus B; Zhu, Lihua J et al. (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol 26:695-701
Meng, Xiangdong; Thibodeau-Beganny, Stacey; Jiang, Tao et al. (2007) Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method. Nucleic Acids Res 35:e81
Meng, Xiangdong; Wolfe, Scot A (2006) Identifying DNA sequences recognized by a transcription factor using a bacterial one-hybrid system. Nat Protoc 1:30-45

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