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A Dissertation Entitled Mapping and CRISPR/Cas9 Gene Editing For A Dissertation entitled Mapping and CRISPR/Cas9 Gene Editing for Identifying Novel Genomic Factors Influencing Blood Pressure by Harshal Waghulde Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences _________________________________________ Bina Joe, PhD, Committee Chair _________________________________________ Guillermo Vazquez, PhD, Committee Member ________________________________________ Kathryn Eisenmann, PhD, Committee Member _________________________________________ Jennifer Hill, PhD, Committee Member _________________________________________ Jiang Tian, PhD, Committee Member _________________________________________ Amanda Bryant-Friedrich, PhD, Dean College of Graduate Studies The University of Toledo August 2016 Copyright 2016, Harshal Bhanudas Waghulde This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Mapping and CRISPR/Cas9 Gene Editing for Identifying Novel Genomic Factors Influencing Blood Pressure by Harshal Waghulde Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences Degree in Doctor of Philosophy Degree in Biomedical Sciences The University of Toledo August 2016 Hypertension is a complex polygenic trait and a significant risk factor for cardiovascular and metabolic diseases. Rodent models serve as tools to identify causal genes for complex traits. This dissertation is comprised of two projects. Project 1 utilizes substitution mapping as an approach to locate blood pressure quantitative trait loci (BP QTLs) on rat chromosome 5 (RNO5) and project 2 utilizes Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated proteins 9 (Cas9) genetic engineering as an approach to explore the physiological function of G-protein coupled estrogen receptor (Gper1) in a rat model of hypertension. Previously, using linkage analysis and substitution mapping, two closely-linked interactive blood pressure quantitative trait loci (QTLs), BP QTL1 and BP QTL2, have been defined within 117894038bp-131853815bp region (RGSC 3.4 version) on rat chromosome 5 (RNO5). This was done by using a series of congenic strains consisting of genomic segments of the Dahl salt-sensitive (S) rat substituted with that of the normotensive Lewis (LEW) rat. Through the construction and characterization of a panel of S.LEW bicongenic strains and corresponding S.LEW monocongenic strains, definitive iii evidence of epistasis (genetic interaction) between BP QTL 1 (7.77Mb) and BP QTL 2 (4.18Mb) has been documented. In order to further map these interacting QTLs, we constructed a new panel of 7 bicongenic strains and monitored their blood pressure by radiotelemetry. The data obtained from these new strains further resolved BP QTL1 from 7.77Mb to 2.93Mb. It was also evident that the QTL2 is not a single QTL, but consists of at least 3 QTLs (2.26Mb, 1.31Mb and 175kb) with contrasting effects on blood pressure. In the second project, we utilized CRISPR/Cas9 genetic engineering approach to study the physiological role of G-protein coupled estrogen receptor (Gper1) in the Dahl-salt sensitive (S) rat. A link between gut microbiota and blood pressure (BP) regulation was previously demonstrated in our laboratory. Gut microbiotal transplantation from Dahl-salt resistant (R) rats into genetically hypertensive Dahl-salt sensitive (S) rats caused an elevation in BP, which was associated with an increase in plasma acetate. Acetate is a short chain fatty acid, which is a known ligand for two of the G-protein coupled receptors, Gpr41 and Olfr78. Deletion of either Gpr41 or Olfr78 is reported to affect BP. Because S and R rats do not have allelic variations of Gpr41 and Olfr78, the observed increased plasma acetate being associated with elevated blood pressure cannot be attributed to these two receptors alone. This led us to hypothesize that yet unknown receptors of acetate exist on the rat genome to regulate BP. To test this hypothesis, we focused on a more recently discovered G-protein coupled estrogen receptor (Gper1) which belongs to the same class of orphan receptors as Gpr41. To completely disrupt Gper1 in S rats, we employed clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) approach with two gRNAs each targeting one end of the rat Gper1 gene. The resultant Gper1-/- rats had significantly iv lower BP and increased vasorelaxation to acetylcholine compared to wild type S rats. Further, to examine whether the presence or absence of Gper1 influence vascular response to short chain fatty acids (acetate, propionate and butyrate), wire myograph studies were conducted using small mesenteric arteries (SMAs). While a rapid contraction effect of acetate and butyrate in phenylephrine pre-contracted arteries were similar, the sustained relaxation following rapid contraction was significantly decreased in vessels from Gper1-/- rats. Because gut microbiota is the source of short chain fatty acids, we conducted microbiotal transplantation studies, data from which demonstrated that the observed BP lowering effect of Gper1-/- was abolished. Collectively, the results point to Gper1 as a novel short chain fatty acid receptor. v This dissertation is dedicated to my beloved parents who have been with me at every step of the way, through good and the bad times, for their unconditional love, guidance, and support, and for raising me to be the person that I am today. I also dedicate my work to my wife for instilling in me the confidence that I am capable of doing anything I put my mind to. Acknowledgements I am grateful to my mentor Dr. Bina Joe for accepting me as a student in her laboratory and giving me exciting projects to work on. It is worth-mentioning that her endless motivation, valuable guidance and meticulousness about every detail helped me develop a genuine interest in the subject and persistently encouraged me to do top notch research. Apart from research, I also learnt many other things from her that would be really helpful to me in the future. I would also like to thank my ex-colleague and best friend Dr. Resmi Pillai for helping me understand the difficult genomic concepts quickly and also for her unwavering support inside as well as outside the lab. I am deeply grateful to my advisory committee members Dr. Vazquez, Dr. Eisenmann, Dr. Hill and Dr. Tian for their valuable suggestions for my research. I further extend my utmost gratitude to our collaborators for their help in the generation of knock out rats. I also thank my past and present lab mates and the Department of Physiology and Pharmacology for all their help and support in this course of my scientific journey. My vote of thanks will be incomplete without mentioning my parents and my brother who have always been a source of encouragement and inspiration for me, as well as my in-laws for their support and understanding. Last but not the least I would like to thank my wife, Priya, for her undying love and support. She is always by my side and helped me find the right direction in every task I took in my hand. vi Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ............................................................................................................ vi Table of Contents .............................................................................................................. vii List of Tables .................................................................................................................... xi List of Figures ................................................................................................................... xii List of Abbreviations .........................................................................................................xv 1 Introduction…. .........................................................................................................1 1.1 Complex traits and genetics ...............................................................................1 1.2 Missing heritability ............................................................................................2 1.2.1 Epistasis ..............................................................................................4 1.2.2 Yet undiscovered variants ...................................................................4 1.3 Hypertension as a complex polygenic trait ........................................................6 1.3.1 Why study the genetics of hypertension? ...........................................7 1.3.2 The rat as a physiological model of hypertension ..............................7 1.4 Genetic methods for analysis of inherited hypertension ....................................9 1.4.1 Genetic linkage analysis .....................................................................9 1.4.1.1 Genetic linkage analysis using rat models ...........................9 1.4.2 Substitution mapping using congenic strains ....................................11 1.4.3 Genome-wide association studies (GWAS)......................................14 vii 1.4.4 Genome editing as a tool to study hypertension ...............................15 1.4.4.1 CRISP/Cas9 .......................................................................17
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