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Strategies for Re-Targeting the Non-LTR Retrotransposon Alu

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Strategies for Re-Targeting the Non-LTR Retrotransposon Alu Strategies for re-targeting the non-LTR retrotransposon Alu AN ABSTRACT SUBMITTED ON THE FIFTH DAY OF SEPTEMBER OF 2017 TO THE DEPARTMENT OF CELL AND MOLECULAR BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE TULANE UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY Catherine M. Ade APPROVED: AstridJ. Engel, David A. Mullin, Ph.D William C. Wimley, Ph.D. ABSTRACT Genetic engineering of biological molecules has provided the ability to create improved tools that have been applied in numerous fields including re- search, agriculture, industrial biotechnology, and medicine. Mobile elements are the perfect platform for developing gene targeting systems in humans, as they are endogenous and capable of mobilizing genetic material. Here, I present data demonstrating the first successful genetic engineering of a human non-LTR ele- ment protein to promote site-specific insertions. I tested two distinct strategies to target Alu insertions to specific sites in the human genome. The first strategy consisted of fusing a site-specific DNA binding domain (DBD) to the L1 ORF2 protein to favor Alu insertions to the target site. Five distinct DNA binding do- mains targeting specific sequences were tested: the Adeno associated virus (AAV) REP proteins, a TAL effector, the Cre recombinase, several zinc fingers, and the catalytically inactive dCas9. The second strategy utilized the CRISPR system, fusing a catalytically active Cas9 protein to an endonuclease deficient L1 ORF2 protein, the targeting abilities provided by a gRNA. I was successful at en- riching insertions of Alu insertions within in 1.2 kb window around the target se- quence by 47 fold by fusing a six-finger zinc finger, ZF4, to the N-terminus of the ORF2 protein. Recovered insertions showed a distinct bias, inserting upstream of the ZF4 target sequence when compared to recovered Alu insertions driven by unfused L1 ORF2. Other DBDs were unable to alter targeting preference of the ORF2 protein. However, the data provided valuable information on the require- ments for designing successful genetic engineering approaches. My findings demonstrate that multiple factors including target site abundance, linker selec- tion, terminus for fusion, size of DNA binding domain, and overall complexity of the system play an important role in designing fusion proteins with targeting ca- pabilities. The data demonstrate that it is possible to redirect insertion prefer- ence of human non-LTR-retroelements. Strategies for re-targeting the non-LTR retrotransposon Alu A DISSERTATION SUBMITTED ON THE FIFTH DAY OF SEPTEMBER OF 2017 TO THE DEPARTMENT OF CELL AND MOLECULAR BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE TULANE UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY rll Catherine M. Ade APPROVED: gel^Ph.D h.D. William C. Wimley, Ph.D. © Copyright by Catherine M. Ade, 2017 All Rights Reserved ACKNOWLEDGEMENTS . I need to first express my gratitude to Astrid Engel, for taking a chance on an awkward CMB student. You have been a great mentor in and outside the lab, giving me countless opportunities to grow and succeed. Without your guidance, wisdom, kindness, and patience, I would not be the person I am today. To the former members of the lab, Dr. Brad Wagstaff and Rebecca Derbes: thank you for training and helping me get through the first few years of graduate school. Without your advice, experience, and humor, this experience would have been quite different. To the members of COMET, especially Dr. Maria Morales in the Deininger lab: thank you for reigning in, dealing with, and sometimes partici- pating in the crazy. All y’all made it an easy decision to join Astrid’s lab. To everyone I consider family- biological relatives, Virginia band geeks, Ohio nerds, and New Orleans comrades- thank you. Thank you for all of your unwa- vering support, lending me your ears and minds. Without you all, this process would not have been as rewarding or entertaining. Finally, extra scritches and cuddles to my fur babies Tazo, Vinnie, Violet, Blue, Frisco, and Boudreaux. You always know how to make me smile. TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………vi LIST OF FIGURES…………………………………………………………………….viii CHAPTER 1. INTRODUCTION……………………………………………………….1 1.1 Mobile elements in the human genome 1.2 LINE-1 1.2.1 LINE-1 Structure 1.2.2 LINE-1 transcription, translation, and expression 1.2.3 LINE-1 replication cycle 1.2.4 Non-Autonomous elements mobilized by LINE-1 1.3 Alu elements 1.3.1 Origin of Alu elements 1.3.2 Alu Mobilization 1.4 Retrotransposons contribute to human diseases 1.4.1 Insertional mutagenesis 1.4.2 Non-allelic Homologous recombination (NAHR) events 1.4.3 Adverse effects from L1 protein expression 1.5 Retroelement activity and expression in cancer 1.6 Environmental influences on mobile element activity 1.7 Host suppression mechanisms i 1.8 Assays to detect retrotransposition events 1.8.1 Alu and L1 retrotransposition assay 1.8.2 Alu and L1 rescue assay 1.8.3 Next-generation sequencing assays to detect new insertions 1.9 Naturally occurring elements exhibit site-specific insertions 1.10 DNA binding domains with targeting capabilities 1.10.1 Adeno-associated viral proteins (AAV) 1.10.2 Cre recombinase 1.10.3 TALENS 1.10.4 Zinc fingers 1.10.5 The CRISPR/Cas9 targeting system 1.11 Engineering mobile elements to promote site-specific insertions CHAPTER 2. MATERIALS & METHODS……………………………59 2.1 Constructs 2.1.A Alu Constructs 2.1.B Creating the fusion proteins: LZ-ORF2 and TZ-ORF2 and Cre- ORF2 (Chapter 3) 2.1.C Creation of the fusion proteins: TAL-ORF2 (Chapter 4) 2.1.D Creation of the fusion proteins: ZF2.17-ORF2,ZF2.18-ORF2, and ZF2.1817-ORF2 (Chapter 5) 2.1.E Creation of the fusion proteins: N-ZF4-ORF2, N-ZF2-ORF2, C-ZF4- ORF2, and C-ZF2-ORF2 with GHL, FL4, and HL4 linkers (Chapters 6) ii 2.1.F Creation of the CRISPR/Cas9 fusion proteins and gRNAs (Chapter 7) 2.2. Retrotransposition, Alu Rescue assay and insert analysis 2.3. Creation of a HeLa-LoxP cell line and HeLa-EGFP cell line. 2.4 Western Blot Analysis CHAPTER 3. The effect of the multimeric DNA binding proteins, TZ and LZ and Cre on redirecting ORF2p targeting capabilities…………………………….84 3.1. Introduction 3.2. Results 3.2.1 Evaluation of the retrotransposition capability of the ORF2-fusion pro- teins. 3.2.2 Expression of the ORF2 fusion proteins 3.2.3 Evaluation of the capability of the ORF2 -fusion proteins to redirect in- sertion preference of the Alu. 3.3. Discussion CHAPTER 4. The Transcription Activator-like Effector: The effect of mono- meric DNA binding domains with a few genomic target sequences on tar- geting capabilities……………………………………………………….………….102 4.1. Introduction 4.2. Results 4.2.1 Evaluation of the retrotransposition capability of the ORF2-fusion pro- teins. 4.2.2 Expression of the TAL-ORF2 fusion protein iii 4.2.3 Evaluation of the capability of the TAL-ORF2 -fusion proteins to redirect insertion preference of the Alu. 4.3 Discussion CHAPTER 5. Zinc finger proteins: The effect of monomeric DNA binding domains with a few genomic target sequences on targeting capabili- ties……………………………………………………………………………..………111 5.1 Introduction 5.2 Results 5.2.1 Evaluation of the retrotransposition capability of the ORF2-fusion pro- teins. 5.2.2 Evaluation of the capability of the ORF2-fusion proteins to redirect inser- tion preference of the Alu. 5.3 Discussion CHAPTER 6. Six-fingered zinc fingers that target multiple sites in the ge- nome enrich for Alu insertions when fused to ORF2.................................117 6.1. Introduction 6.2. Results 6.2.1 Evaluation of the retrotransposition capability of the ORF2-fusion pro- teins. 6.2.2 Expression of the ORF2 fusion proteins 6.2.3 Evaluation of the capability of the ORF2 -fusion proteins to redirect in- sertion preference of the Alu. 6.2.4 Insertion bias observed due to availability of endo site 6.3. Discussion iv CHAPTER 7. Adapting the CRISPR system to the ORF2 protein in order to target Alu insertions………………………………………………………………..142 7.1 Introduction 7.2 Results 7.2.1 Evaluation of the Cas9 targeting capability of the Cas9-ORF2-fusion proteins. 7.2.2 Evaluation of the retrotransposition capability of the functional Cas9- endonuclease defective ORF2-fusion proteins. 7.2.3 Evaluation of the capability of the functional Cas9-endonuclease defec- tive ORF2-fusion proteins to redirect insertion preference of Alu. 7.2.4 Evaluation of the capability of the dCas9-ORF2 (defective Cas9- functional ORF2)-fusion proteins to redirect insertion preferences of Alu. 7.2.5 Using an MS2-ORF2 fusion protein to increase the interaction a MS2- gRNA-Cas9 complex to increase efficiency of targeting Alu insertions 7.2.6 Re-designing the A-tail of the Alu Rescue Cassette to promote se- quence homology between a specific genomic target and Alu insert. 7.3: Discussion CHAPTER 8. CONCLUSIONS……………………………………………...……..168 BIBLIOGRAPHY……………………………………………………………………..174 APPENDIX……………………………………………………………………………203 BIOGRAPHY…………………………………………………………………………264 v LIST OF TABLES Table 1. Relative retrotransposition rates of Alu when driven by ORF2 or by the indicated fusion protein………………………………………………………………..93 Table 2. Relative retrotransposition rates of Alu when driven by ORF2 or by N- terminally fused TAL effector………………………………………………………. 106 Table 3. Two potential Alu targeting events when Alu is driven by TAL- ORF2…………………………………………………………………………………..108 Table 4. Relative retrotransposition rates of Alu when driven by ORF2 or by N- terminally fused EGFP targeting zinc fingers…………………………………… 114 Table 5. Relative retrotransposition rates of Alu when driven by ORF2 or by the N-ZF4-ORF2 fusion protein…………………………………………………………119 Table 6. Recovered Alu Retrotransposition events driven by the ORF2 and the N-ZF4-ORF2 fusion construct………………………………………………………122 Table 7.
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