Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2013 Computational and experimental analysis of TAL effector-DNA binding Erin Lynn Doyle Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Agricultural Science Commons, Agriculture Commons, Bioinformatics Commons, Biology Commons, and the Plant Pathology Commons Recommended Citation Doyle, Erin Lynn, "Computational and experimental analysis of TAL effector-DNA binding" (2013). Graduate Theses and Dissertations. 13132. https://lib.dr.iastate.edu/etd/13132 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Computational and experimental analysis of TAL effector-DNA binding by Erin Lynn Doyle A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Bioinformatics and Computational Biology Program of Study Committee: Adam J. Bogdanove, Co-Major Professor Daniel Nettleton, Co-Major Professor Susan Lamont Leonor Leandro Steve Whitham Iowa State University Ames, Iowa 2013 Copyright © Erin Lynn Doyle, 2013. All rights reserved. ii TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................... vii LIST OF TABLES ....................................................................................................................... ix ABSTRACT ................................................................................................................................. x CHAPTER 1. GENERAL INTRODUCTION ................................................................................ 1 Abbreviations .......................................................................................................................... 1 Introduction ............................................................................................................................ 1 Rationale ................................................................................................................................ 2 Dissertation Organization ....................................................................................................... 3 References ............................................................................................................................. 5 CHAPTER 2. THE EFFECTS OF TAL EFFECTORS ON CELL BIOLOGY .............................. 7 Abstract .................................................................................................................................. 7 Introduction ............................................................................................................................ 7 TAL effector structure and function ......................................................................................... 8 The natural role of TAL effectors ...........................................................................................11 TAL effectors and improving plant disease resistance ...........................................................13 TAL effectors as customizable DNA targeting proteins ..........................................................14 Fusions for targeted gene activation and repression ..........................................................14 TAL effector fusions for genome editing .............................................................................15 Design and construction .....................................................................................................17 Future challenges ..................................................................................................................17 References ............................................................................................................................19 CHAPTER 3. EFFICIENT DESIGN AND ASSEMBLY OF CUSTOM TALEN AND OTHER TAL EFFECTOR-BASED CONSTRUCTS FOR DNA TARGETING ..........................................25 Abstract .................................................................................................................................25 Introduction ...........................................................................................................................26 Materials and Methods ..........................................................................................................29 Protocol for assembly of custom TALEN, TAL effector or TAL effector fusion-ready constructs ..........................................................................................................................29 Construction of module, last repeat, array, and backbone plasmids ...................................31 iii Software to identify candidate TALEN target sites ..............................................................33 Testing TALEN function in yeast ........................................................................................34 Expression of custom TALENs in human cells and Arabidopsis protoplasts and detection of site-specific mutations.....................................................................................35 Expression of a custom TAL effector in Xanthomonas and in planta activity assay ............36 Results ..................................................................................................................................36 Efficient assembly of custom repeat arrays into TALEN and other TAL effector-based constructs ..........................................................................................................................36 Guidelines and software for TALEN site selection and repeat array design. .......................37 Activity of custom TALENs in a yeast-based DNA cleavage assay ....................................39 Targeted mutagenesis in human cells and Arabidopsis protoplasts using custom TALENs .............................................................................................................................39 Replication of AvrHah1 TAL effector activity with a Golden Gate assembled clone ............40 Discussion .............................................................................................................................41 Acknowledgements ...............................................................................................................44 Funding .................................................................................................................................44 References ............................................................................................................................45 Supplementary Material .........................................................................................................47 CHAPTER 4. TAL EFFECTOR-NUCLEOTIDE TARGETER (TALE-NT) 2.0: TOOLS FOR TAL EFFECTOR DESIGN AND TARGET PREDICTION ..........................................................53 Abstract .................................................................................................................................53 Introduction ...........................................................................................................................53 Software and Algorithms .......................................................................................................56 Programming .....................................................................................................................56 Tools for designing custom TALENs and TAL effectors .....................................................56 A tool for identifying candidate TAL effector targets ...........................................................56 Web Interface ........................................................................................................................57 Design and general features ..............................................................................................57 TALEN Targeter .................................................................................................................58 TAL Effector Targeter.........................................................................................................59 Target Finder .....................................................................................................................59 Conclusion ............................................................................................................................61 Acknowledgements ...............................................................................................................62 iv Funding .................................................................................................................................62 References ............................................................................................................................62 Supplementary Material .........................................................................................................64 The Target Finder scoring function ....................................................................................64 References