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MULTIPLEX GENOME EDITING and GENE REGULATION in EUKARYOTIC CELLS by ZEHUA BAO DISSERTATION Submitted in Partial Fulfillment of T

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MULTIPLEX GENOME EDITING and GENE REGULATION in EUKARYOTIC CELLS by ZEHUA BAO DISSERTATION Submitted in Partial Fulfillment of T MULTIPLEX GENOME EDITING AND GENE REGULATION IN EUKARYOTIC CELLS BY ZEHUA BAO DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2017 Urbana, Illinois Doctoral Committee: Professor Huimin Zhao, Chair Professor James H. Morrissey Professor Jie Chen Assistant Professor Kai Zhang ABSTRACT Biological functions of a cell are encoded within its genome and regulated through complex genetic networks. Direct manipulation of the genome and the transcriptome in multiplex enables study of gene-gene interactions and rapid creation of industrially or therapeutically useful cells. Such manipulations, including genome editing and epigenetic regulation of gene expression, rely on a group of precise DNA-binding proteins, which include zinc finger proteins (ZFPs), transcription activator like effectors (TALEs), and clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9). ZFPs and TALEs rely on protein domain shuffling for targeting different genomic loci, which can be laborious and expensive. Instead, CRISPR/Cas9 is guided by a 20 bp ribonucleotide, which can be synthesized in multiplex rapidly and cost-effectively. This dissertation describes my efforts in engineering the CRISPR/Cas9 system for multiplex genome editing and gene regulation in eukaryotic cells. The first part of this dissertation (Chapters 2 and 3) focuses on genome editing in Saccharomyces cerevisiae. S. cerevisiae, also known as the baker’s yeast, is an important model eukaryotic organism for basic research, as well as a preferred industrial host for production of biofuels, pharmaceuticals and fine chemicals. I developed a Homology-Integrated CRISPR/Cas9 (HI- CRISPR) system for concurrent disruption of up to three genes in S. cerevisiae. This system enabled rapid and efficient generation of yeast strains with multiple mutations and should prove useful for studying yeast gene functions. Based on the design of the HI-CRISPR system, I further developed a CRISPR/Cas9 and homology-directed repair assisted genome-scale engineering (CHAnGE) system for engineering and directed evolution of S. cerevisiae strains. Using the ii CHAnGE system, I identified both known and novel genes susceptible to acetic acid or furfural toxicity. Finally, I demonstrated one-step, high efficiency in vivo site directed mutagenesis in S. cerevisiae for introducing designed mutations into native chromosomal contexts. The second part of this dissertation (Chapters 4 and 5) focuses on ligand-inducible, multiplex gene regulation in mammalian cells. Ligand-inducible gene regulation enables temporal control of gene functions, which is necessary in interrogating dynamic gene regulatory networks. However, temporal control is currently limited on a single gene level. I developed chemically induced transcription activators by combining orthogonal CRISPR/Cas9 systems and chemically induced dimerizing proteins. In HEK293T cells, these transcription activators exerted simultaneous activation of multiple genes and orthogonal regulation of different genes in a ligand-dependent manner with minimal background. As proof of concept, I attempted to convert mouse embryonic fibroblasts to neuronal cells using chemically induced CRISPR/Cas9 activators. iii To Father, Mother, and Grandmother iv ACKNOWLEDGMENTS This dissertation would not have been possible without the support of many people. Many thanks to my advisor, Dr. Huimin Zhao, who offered me financial and intellectual support throughout the process of completing this dissertation. Without his support, it would be difficult for me to concentrate on the various research projects that I have tried in the past five years. Thanks to my doctoral committee members, Dr. James H. Morrissey, Dr. Jie Chen, and Dr. Kai Zhang, who supported me with my research and kindly served as my references when I looked for a postdoctoral position. Thanks to Dr. Satish K. Nair and Dr. Paul J. Hergenrother for serving on the committee for my qualifying examination. I would also like to give thanks to my former doctoral committee member, Dr. Colin A. Wraight, who passed away in 2014 after a brave fight against cancer. Thanks to Dr. Yi Lu and Dr. Susan A. Martinis, who took me as a rotation student when I first started my Ph.D. study. Thanks to the UIUC Block Grant Fellowship for supporting my rotations. Thanks to the Department of Biochemistry, the School of Molecular and Cellular Biology, and the Graduate School at UIUC for awarding me travel grants to conferences for professional development. Thanks to all the past and current members of Zhao group for their valuable discussions and encouragements. In particular, I would like to thank Dr. Ning Sun, Dr. Han Xiao, Dr. Jing Liang, Dr. Tong Si, Dr. Sijin Li, and Dr. Zhanar Abil for mentoring and helping me with my experimental skills and research projects. Many thanks to Mohammad (Sam) HamediRad, Lu Zhang, Surbhi Jain, Ran Chao, Ipek Tasan, Xiong Xiong, Dr. Zhen Kang, Dr. Yi Yu, Dr. Ryan E. Cobb, and Pu Xue for collaborating on publications or giving helpful suggestions. Thanks to v Dr. Yunzi Luo, Dr. Hua Huang, Dr. Meng Wang and Dr. Yongbo Yuan for organizing and participating in extracurricular activities that made my Ph.D. life interesting. I would like to give special thanks to my undergraduate research assistant, Valerie Jaroenpuntaruk, who helped me with essentially all of my research projects and allowed me time to do more critical thinking. I would like to thank Dr. Alvaro G. Hernandez and Chris L. Wright at Roy J. Carver Biotechnology Center for their very kind help with next generation sequencing, Dr. Jenny Drnevich Zadeh at Roy J. Carver Biotechnology Center for help with biological computing, and Dr. Sandra K. McMasters at UIUC Cell Media Facility for assistance with media preparation. This dissertation would also not have been possible without the research materials provided by Dr. Feng Zhang (Massachusetts Institute of Technology), Dr. Jennifer A. Doudna (University of California, Berkeley), Dr. Charles A. Gersbach (Duke University), Dr. Takanari Inoue (Johns Hopkins University), and Dr. Tasuku Ueno (The University of Tokyo). I would like to thank Dr. Hanchao Zhao, Dr. Pengfei Yu, Dr. Zhongyi Li, Dr. Li Li, Dr. Ruizhi Li, Weiying Zhang, Binyue Hou, Dr. Yueyi Liu, Shijia Tang, Dr. Chunmei Gu for their friendship. I would like to thank Dr. Sam Lee for serving as my leadership coach at the Illinois Leadership Center. I would like to thank my girlfriend, Dr. Jie Zhu, who endured the hardest time with me. I would like to thank my brother, Wenfeng Bao, who mentored and supported me to be an independent person. Finally and most importantly, I would like to thank my parents and my grandmother for their continuous support and unconditional love. vi TABLE OF CONTENTS Chapter 1. Introduction .............................................................................................................. 1 1.1 CRISPR/Cas9 as an RNA-guided DNA-binding Protein ........................................................ 1 1.2 Multiplex Targeting by CRISPR/Cas9 .................................................................................... 5 1.3 Genome Editing Using CRISPR/Cas9 .................................................................................... 6 1.4 The Concepts of Multiplex Genome Engineering and Genome-scale Engineering ................ 8 1.5 Gene Regulation Using dCas9 Activators ............................................................................. 10 1.6 Inducible Gene Regulation .................................................................................................... 11 1.7 Overview of this Dissertation ................................................................................................ 12 1.8 References ............................................................................................................................. 13 Chapter 2. Multiplex Genome Editing in Saccharomyces cerevisiae .................................... 19 2.1 Introduction ........................................................................................................................... 19 2.2 Results ................................................................................................................................... 21 2.2.1 Design and Optimization of a Homology-Integrated CRISPR/Cas9 System for Efficient Gene Disruption ......................................................................................................................... 21 2.2.2 One-step Triple Gene Disruption in Saccharomyces cerevisiae ..................................... 31 2.2.3 One-step Disruption of More Than Three Genes ........................................................... 36 2.3 Discussion .............................................................................................................................. 36 2.4 Materials and Methods .......................................................................................................... 40 2.4.1 Strains and Media ........................................................................................................... 40 2.4.2 Cloning of DNA Constructs ............................................................................................ 41 2.4.3 Yeast Transformation .....................................................................................................
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