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Zinc Finger Nucleases (Zfns) and Transcription Activator-Like Effector Nucleases (Talens) Deepak Reyon Iowa State University

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Zinc Finger Nucleases (Zfns) and Transcription Activator-Like Effector Nucleases (Talens) Deepak Reyon Iowa State University Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2011 Genome engineering using DNA-binding proteins: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) Deepak Reyon Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Bioinformatics Commons, Computational Biology Commons, and the Genomics Commons Recommended Citation Reyon, Deepak, "Genome engineering using DNA-binding proteins: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)" (2011). Graduate Theses and Dissertations. 14745. https://lib.dr.iastate.edu/etd/14745 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]. Genome engineering using DNA-binding proteins: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) by Deepak Reyon 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: Drena Dobbs, Co-Major Professor Edward Yu, Co-Major Professor Vasant Honavar Amy Andreotti Gaya Amarasinghe Iowa State University Ames, Iowa 2011 Copyright © Deepak Reyon, 2011. All rights reserved. ii TABLE OF CONTENTS LIST OF FIGURES…………………………………………………………….iiiv LIST OF TABLES ................................................................................................. ix ABSTRACT ............................................................................................................ x CHAPTER 1. GENERAL INTRODUCTION ........................................................ 1 BACKGROUND ................................................................................................ 1 METHODS FOR ASSEMBLING TALENs. ...................................................... 7 OVERALL GOAL AND RESEARCH AIMS ...................................................11 DISSERTATION ORGANIZATION ................................................................ 12 REFERENCES ................................................................................................. 14 CHAPTER 2. PREDICTING SUCCESS OF OLIGOMERIZED POOL ENGINEERING (OPEN) FOR ZINC FINGER TARGET SITE SEQUENCES .............................................................................................. 16 ABSTRACT ...................................................................................................... 16 Background: .................................................................................................. 16 Results: .......................................................................................................... 16 Conclusion: ................................................................................................... 17 BACKGROUND .............................................................................................. 17 RESULTS .......................................................................................................... 19 DISCUSSION ................................................................................................... 25 CONCLUSION ................................................................................................. 27 METHODS ....................................................................................................... 28 Definition of active and inactive ZFP target sites based on B2H assays ...... 28 Datasets of experimentally validated ZFP-target sites .................................. 28 iii Machine learning classifiers ......................................................................... 29 Target site sequence encoding ....................................................................... 30 Classification performance measures ........................................................... 30 Confidence Score .......................................................................................... 31 COMPETING INTERESTS ............................................................................. 31 AUTHORS’ CONTRIBUTIONS ..................................................................... 31 ACKNOWLEDGEMENTS .............................................................................. 32 REFERENCES ................................................................................................. 32 CHAPTER 3. ZFNGENOME: A COMPREHENSIVE RESOURCE FOR LOCATING ZINC FINGER NUCLEASE TARGET SITES IN MODEL ORGANISMS ........................................................................................ 43 ABSTRACT ...................................................................................................... 43 Background ................................................................................................... 43 Description .................................................................................................... 43 Conclusions ................................................................................................... 44 BACKGROUND .............................................................................................. 44 CONSTRUCTION AND CONTENT............................................................... 47 Resources available in ZFNGenome ............................................................ 51 DISCUSSION ................................................................................................... 53 Related Resources ......................................................................................... 55 Planned future development ......................................................................... 56 CONCLUSIONS............................................................................................... 56 AVAILABILITY AND REQUIREMENTS ...................................................... 57 LIST OF ABBREVIATIONS USED ................................................................ 57 iv AUTHORS' CONTRIBUTIONS ...................................................................... 57 ACKNOWLEDGEMENTS .............................................................................. 57 REFERENCES ................................................................................................. 58 CHAPTER 4. TARGETED GENE DISRUPTION IN SOMATIC ZEBRAFISH CELLS USING ENGINEERED TALENS .................................... 61 To the Editor: .................................................................................................... 61 ACKNOWLEDGEMENTS .............................................................................. 65 CONFLICT OF INTEREST STATEMENT: .................................................... 65 REFERENCES ................................................................................................. 65 CHAPTER 5. USER-FRIENDLY PROTOCOL AND SOFTWARE FOR RAPID ENGINEERING OF DESIGNER TALE NUCLEASES (TALENS) ...... 67 ABSTRACT ...................................................................................................... 67 INTRODUCTION ............................................................................................ 67 OVERVIEW OF THE PROCEDURE .............................................................. 70 Identifying TALEN target sites using the ZiFiT Targeter program: ............. 70 Assembly of plasmid DNA encoding TALE repeat arrays: .......................... 70 Cloning of DNA encoding TALE repeat arrays into a TALEN expression vector: ......................................................................................... 70 MATERIALS .................................................................................................... 71 REAGENTS .................................................................................................. 71 EQUIPMENT ............................................................................................... 72 PROCEDURE ................................................................................................... 72 Identification of potential TALEN target sites using web-based ZiFiT Targeter software ................................................................................ 72 v Construction of TAL Arrays .......................................................................... 73 Cloning TAL arrays into the nuclease backbone .......................................... 77 ANTICIPATED RESULTS ............................................................................... 80 AUTHOR CONTRIBUTIONS ......................................................................... 81 ACKNOWLEDGMENTS ................................................................................ 81 REFERENCES ................................................................................................. 81 CHAPTER 6. GENERAL CONCLUSIONS & FUTURE DIRECTIONS .......... 83 CONTRIBUTIONS OF THIS DISSERTATION .............................................. 85 FUTURE STUDIES.......................................................................................... 87 REFERENCES ................................................................................................. 91 APPENDIX A. SELECTION-FREE ZINC-FINGER-NUCLEASE
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