TAL effectors are a class of proteins used by bacterial pathogens of plants to directly modulate host gene expression. They bind to effector-specific host DNA sequences, and this specificity is governed by a one-to- one correspondence between a variable pair of residues in each repeat and nucleotides that make up the target DNA sequences. TAL effectors with new DNA sequence specificities can be created by assembling the appropriate repeats in the necessary order in the DNA binding domain. Further, the fusion of native or tailored TAL effector DNA binding domains to the restriction endonuclease FokI creates targeted double-strand DNA breaks at predicted sequences. These properties indicate that TAL effectors hold much promise as customizable DNA binding scaffolds for targeted gene regulation and genetic modification. To realize this potential, this project aims to 1) determine the basis for TAL effector DNA binding affinity and specificity, 2) enhance targeting capacity, and 3) implement and assess custom TAL effectors and TAL effector nucleases for genome manipulation.
The first aim will be accomplished by determining the contributions of associations of individual TAL effector repeats with target nucleotides to overall binding affinity and specificity and whether the contributions are cooperative or additive. Further, biophysical and crystallographic structure analysis will be performed for distinct TAL effectors bound to their DNA targets.
The second aim will be approached by incorporating repeats with novel residue pairs that have potential for enhanced nucleotide affinity and specificity and by making modifications outside the repeat region that are predicted to relax the requirement for thymine at position -1 of each binding site. Finally, aim 3 will be achieved by using TAL effectors as custom transcription factors and TALENs as reagents for gene disruption and gene editing to determine efficacy, efficiency, and fidelity. These experiments will be conducted in both Arabidopsis and human cells to determine breadth of applicability and to take advantage of the strengths of each model to assess off-target effects.

Public Health Relevance

The proposed project will increase our understanding of DNA targeting by TAL effectors for many possible applications in human as well as plant and animal health. Of particular relevance to human health, genome modification strategies are increasingly being pursued in gene therapy, and the proposed research will establish the therapeutic potential of TAL effector based reagents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098861-04
Application #
8655549
Study Section
Therapeutic Approaches to Genetic Diseases (TAG)
Program Officer
Sledjeski, Darren D
Project Start
2011-09-01
Project End
2015-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
4
Fiscal Year
2014
Total Cost
$506,629
Indirect Cost
$70,608
Name
Iowa State University
Department
Other Basic Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
005309844
City
Ames
State
IA
Country
United States
Zip Code
50011
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Boissel, Sandrine; Jarjour, Jordan; Astrakhan, Alexander et al. (2014) megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Nucleic Acids Res 42:2591-601
Bogdanove, Adam J (2014) Principles and applications of TAL effectors for plant physiology and metabolism. Curr Opin Plant Biol 19:99-104
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Kleinstiver, Benjamin P; Wang, Li; Wolfs, Jason M et al. (2014) The I-TevI nuclease and linker domains contribute to the specificity of monomeric TALENs. G3 (Bethesda) 4:1155-65
Cernadas, Raul A; Doyle, Erin L; NiƱo-Liu, David O et al. (2014) Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene. PLoS Pathog 10:e1003972
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Zhang, Yong; Zhang, Feng; Li, Xiaohong et al. (2013) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol 161:20-7
Nyquist, Michael D; Li, Yingming; Hwang, Tae Hyun et al. (2013) TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer. Proc Natl Acad Sci U S A 110:17492-7

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