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Towards in Vivo Editing of the Human Microbiome

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Towards in Vivo Editing of the Human Microbiome Towards in vivo editing of the human microbiome by Stephanie J. Yaung S.B. Biological Engineering Massachusetts Institute of Technology, 2010 S.B. Management Science Massachusetts Institute of Technology, 2010 Submitted to the Harvard-MIT Division of Health Sciences and Technology in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Medical Engineering and Medical Physics at the Massachusetts Institute of Technology June 2015 © 2015 Massachusetts Institute of Technology. All rights reserved. Signature of Author: _____________________________________________________________ Harvard-MIT Division of Health Sciences and Technology May 8, 2015 Certified by: ___________________________________________________________________ George M. Church, PhD Professor of Genetics Harvard Medical School Thesis Supervisor Accepted by: ___________________________________________________________________ Emery N. Brown, MD, PhD Professor of Computational Neuroscience and Health Sciences and Technology Director, Harvard-MIT Program in Health Sciences and Technology 2 Towards in vivo editing of the human microbiome by Stephanie J. Yaung Submitted to the Harvard-MIT Division of Health Sciences and Technology on May 8, 2015 in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Engineering and Medical Physics Abstract The human microbiota consists of 100 trillion microbial cells that naturally inhabit the body and harbors a rich reservoir of genetic elements collectively called the microbiome. Efforts based on metagenomic sequencing of microbiomes associated with healthy and diseased individuals have revealed vast effects of microbiota on human health. However, compared to the expanding amount of sequence data, little is known about the function of these microbes and their genes. Furthermore, current clinical approaches to modify the microbiota face several challenges, including colonization resistance in competitive environments such as the gut, and imprecise ecological perturbations using antibiotics and fecal transplants. The fundamental objective of this research is to develop safe methods to genetically edit the microbiome in vivo to promote human health. The abilities to introduce commensally fit strains and to control specificity of microbial modulations are critical steps towards ecological engineering of healthy microbiota. This thesis describes strategies to investigate, propagate, and ultimately engineer desired functions in microbiota. In particular, we developed a temporal functional metagenomics method to identify genes that improved microbial fitness in the mammalian gut in vivo. We also built foundational tools for delivering genetic elements and immunizing endogenous microbiota against acquiring antibiotic resistance and toxins. In addition to leveraging bacterial conjugation and the prokaryotic defense system CRISPR-Cas9, we employed bacteriophages for depleting native strains to empty the niche for an engineered version. Our work enables applications in engineering probiotic strains with augmented fitness and anti-pathogenesis properties, tempering host autoimmunity, and combating hospital-acquired infections and enteric diseases. Thesis Supervisor: George M. Church, PhD Title: Professor of Genetics, Harvard Medical School 3 Dedication To my parents, Fangling Chang and Alan Tsu-I Yaung To my grandparents, Chin-Wan Chang and Chin-Pin Chiu To my uncle Fred Fang-Jen Chang, aunt Connie Tze-Mei Chen, and cousins Angela Chang and Bora Chang for their endless love, encouragement, and support. 4 Acknowledgements The first thing I learned in graduate school was that science is done by people. Science may be the pursuit of knowledge and objective truth, but the process of research and invention is really a human endeavor. Therefore, I would like to thank the people who made this work possible, especially those who gave me honest counsel, valuable guidance, and good company during the ups and downs. First, I am indebted to my research advisor George Church for his inspiration, kindness, and insightful advice throughout my time in graduate school. I am grateful for the opportunity to be a part of the uniquely inventive environment that he has fostered by bringing together diverse people and resources. I would like to thank Eric Alm and Matt Waldor for serving on my thesis committee and providing generous support and constructive feedback. My graduate work would not exist without remarkable collaborators, including Harris Wang, Kevin Esvelt, Georg Gerber, Lynn Bry, and Matt Waldor. I sincerely thank them for the exceptional training and stimulating discussions. I am incredibly honored to have had the opportunity to work closely with Harris and Kevin, who are amazing scientists and thoughtful mentors. When I first joined the Church lab, Harris let me tag along on his new microbiome adventures. Kevin also saw potential and recruited me to be a coconspirator in some of his immense undertakings. They truly helped me launch and advance my graduate research career. I am also deeply grateful to Georg and Matt, who made time to meet with me and served as unofficial advisors in several aspects of my thesis research. I was also fortunate to work with skilled colleagues in these collaborations, including Jonathan Braff, Rose Deng, and Ning Li, who contributed significantly to portions of this work. I would also like to thank Pooja Jethani, a MIT UROP student, and Takahiro Yokoi, a visiting graduate student, for their hard work and contributions. In addition to crucial human capital, I must acknowledge the financial backing for my work, sponsored by the DOE, NIH, DARPA, and NSF through grants awarded to George, Harris, Georg, and Kevin, as well as the Wyss Institute. I am also thankful for support from the NSF Graduate Research Fellowship Program and the MIT Neurometrix Presidential Fellowship. At a place like Harvard or MIT, having brainpower and willpower can become so commonplace that perhaps what stands out more is sincere compassion. In addition to my wonderful scientific collaborators, many others have contributed positively to my time in graduate school and have shown me great kindness. To past and present Church lab members, I will treasure the banter and profound and candid conversations we shared. In particular, I would like to thank Alex Chavez and Jon Scheiman for being like big brothers to me and keeping me grounded; Noah Davidsohn and Noah Taylor for being easygoing neighbors and a calm force when times were hectic; Dan Goodman and Vatsan Raman for always being supportive and caring; and Susan Byrne, Su Vora, Andie Smidler, Michael Napolitano, Alex Garruss, Nikolai 5 Eroshenko, Prashant Mali, Jay Lee, Sri Kosuri, Eric Kelsic, Di Zhang, Mike Chou, John Aach, Sara Vassallo, Mike Mee, Henry Lee, Marc Lajoie, James DiCarlo, Xavier Rios, Alex Ng, Javier Fernández Juárez, Reza Kalhor, Marc Güell, Mike Sismour, Justin Feng, Anik Debnath, George Chao, Ben Stranges, Eswar Iyer, Raj Chari, Fred Vigneault, Sven Dietz, Bobby Dhadwar, Yu Wang, Noah Donoghue, Adam Marblestone, Evan Daugharthy, Uri Laserson, Adrian Briggs, Julie Norville, Barry Wanner, Dima Ter-Ovanesyan, Matthieu Landon, Jun Teramoto, Wei Leong Chew, Jamie Rogers, Nathan Johns, Chris Guzman, Joe Negri, Mirko Palla, Gleb Kuznetsov, Mingjie Dai, Margo Monroe, Joyce Yang, Madeline Ball, Arthur Sun, Jun Li, Luhan Yang, Po-yi Huang, Alex Hernandez-Siegel, Seth Shipman, Venky Soundararajan, Ido Bachelet, Chao Li, Rigel Chan, Tara Gianoulis, Josh Mosberg, Dan Mandell, Danny Levner, Charles Fracchia, Roger Conturie, Joe Davis, Yveta Masar, Meghan Radden, Laura Glass, Stan Fields, Frank Poelwijk, and several others for lending a hand, offering input, and adding colorful memories to my Church lab experience. Being a part of the Harvard-MIT HST program, HMS Genetics, and the Wyss Institute, I would like to express my gratitude to those who kept everything running as smoothly as possible. At HST, I thank my academic advisor Richard Cohen for making sure I was on track throughout my graduate program, and Julie Greenberg, Laurie Ward, Traci Anderson, and Joe Stein for all their work behind the scenes. I would like to acknowledge Vonda Shannon, Ella Sexton, Scott Blackwell, Heidi Turcotte, and Terri Broderick at HMS Genetics, Kelly Seary at the HIM animal facility, and several individuals at the Wyss, including Susan Kelly, Jeanne Nisbet, Martin Montoya-Zavala, Joel Rivera-Cardona, Angel Velarde, Ngawang Sherpa, Amanda Graveline, Andyna Vernet, Matt Balestrieri, Rich Terry, Brian Turczyk, Marcelle Tuttle, and Ben Pruitt for their assistance. I also thank the Harvard MSI for providing a warm community for students in the microbial sciences. Furthermore, I am grateful to all the faculty, teaching staff, and fellow classmates in my HST courses, from Pathology to ICM at Mount Auburn Hospital, for the memorable learning experience and incomparable exposure to clinical medicine. I am especially thankful to fellow students in and outside of HST who were part of our weekly lunches, particularly George Xu, Luvena Ong, Hanlin Tang, James Kath, Thomas Graham, and Luis Barrera, with guest appearances by Sandeep Koshy, Vikram Juneja, and Helen Hou; I came to think of it as a support group that helped preserve our well-being. I owe much gratitude to Luvena for helping me survive graduate school; I am glad we joined neighboring labs at the Wyss and could keep each other afloat during the high tides, though I suspect I benefited much more, at least
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