TARGETING ZEBRAFISH HAIR BUNDLE- RELATED GENES USING CRISPR/CAS9 by HAIMENG BAI Submitted in partial fulfillment of the requirements for the degree of Master of Science Thesis Advisor: Brian M. McDermott Jr., Ph.D. Department of Biology CASE WESTERN RESERVE UNIVERSITY August, 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Haimeng Bai candidate for the Master of Science degree*. Roy Ritzmann, Ph.D. Chair of Committee Brian M. McDermott, Ph.D. Ruben Stepanyan, Ph.D. Audrey Lynn, Ph.D. (date) 5/24/2017 *We also certify that written approval has been obtained for any proprietary material contained therein. 1 Table of Contents Abstract .................................................................................................................... 12 Chapter 1: Background Information ........................................................................... 13 Hearing in Vertebrates ......................................................................................................... 14 Hair Cell ................................................................................................................................ 15 Hair Bundle ........................................................................................................................... 19 Using Zebrafish as a Model Organism .................................................................................. 21 Chapter 2: Targeting Zebrafish Hair Bundle-Related Genes Using CRISPR/Cas9 .......... 23 Introduction ....................................................................................................................... 24 MET Complex/Channel ......................................................................................................... 24 TMC Protein Family .............................................................................................................. 26 LHFPL5 (TMHS) ..................................................................................................................... 30 MACF1 .................................................................................................................................. 31 Materials and Methods ...................................................................................................... 36 Results ............................................................................................................................... 44 CRISPR/Cas9 induced mutation of tmc5 in zebrafish ............................................................ 44 CRISPR/Cas9 induced mutation of lhfpl5a in zebrafish ......................................................... 49 CRISPR/Cas9 induced mutation of tmc6b in zebrafish .......................................................... 52 CRISPR/Cas9 induced mutation of macf1a in zebrafish ........................................................ 56 Discussion .......................................................................................................................... 60 Chapter 3: Generation of transgenic zebrafish line expressing dendra2 tagged β-actin in hair cells ................................................................................................................ 63 2 Introduction ................................................................................................................ 64 β-actin in Hair Cell ......................................................................................................... 64 Dendra2 ........................................................................................................................ 66 Materials and Methods ............................................................................................... 67 Plasmid Preparation and Microinjection ...................................................................... 67 Screening for Positive Fish ............................................................................................ 68 Photoconversion ........................................................................................................... 68 Results ........................................................................................................................ 69 Discussion ................................................................................................................... 71 References .......................................................................................................... 74 3 Acknowledgements First and for most, I would like to thank my Master's advisor, Dr. Brian M. McDermott for his education, continuous support, and caring during the past two years. He patiently encouraged me when I was low and fully supported me by giving me intellectual freedom in my projects. His guidance helped me at all times during research. From him, I experienced the biggest passion for science. Besides work, he is also a thoughtful friend to me who cares about me and is always ready to help. I would like to thank the rest of my thesis committee: Dr. Roy Ritzmann, Dr. Audrey Lynn, and Dr. Ruben Stepanyan for their support, guidance, and education. I am also very thankful to Dr. Jessica Fox and Ms. Julia Brown-Allen for all their help with my future career. My thanks also go to all the members of McDermott lab, Carol Fernando, Hoa Nguyen, Zongwei Chen, Shaoyuan Zhu, Robin Woods Davis, Shengxuan Wang, Shenyu Sun, Jun Wang, Danyang Zhu, and Kayla Kindig, and former lab member Dr. Philsang Hwang and Nilay Gupta, especially for all their valuable advice and training. My particular thanks to my parents for their love and support, and to my girlfriend, Zhuo Chen, for her unwavering companionship and love. I would like to especially thank God, who saved me and provided me this chance to be trained as a Master of Science student out of his wonderful work. For his power, I can 4 finish this journey and overcome the difficulties in my projects, and for his love, I can walk in the light and rejoice. 5 Dedication This thesis is dedicated to the God. "For thine is the kingdom, and the power, and the glory, forever. Amen." 6 List of Tables Table 1. ............................................................................................................................. 39 Table 2. ............................................................................................................................. 41 Table 3. ............................................................................................................................. 42 Table 4. ............................................................................................................................. 61 7 List of Figures Figure 1.1. ......................................................................................................................... 15 Figure 1.2. ......................................................................................................................... 16 Figure 1.3. ......................................................................................................................... 18 Figure 1.4. ......................................................................................................................... 20 Figure 1.5. ......................................................................................................................... 22 Figure 2.1. ......................................................................................................................... 25 Figure 2.2 ......................................................................................................................... 28 Figure 2.3 ......................................................................................................................... 30 Figure 2.4 ......................................................................................................................... 32 Figure 2.5 ......................................................................................................................... 33 Figure 2.6 ......................................................................................................................... 35 Figure 2.7. ......................................................................................................................... 37 Figure 2.8. ........................................................................................................................ 45 Figure 2.9. ........................................................................................................................ 47 Figure 2.10. ....................................................................................................................... 49 Figure 2.11 ....................................................................................................................... 50 Figure 2.12 ....................................................................................................................... 52 Figure 2.13. ....................................................................................................................... 52 Figure 2.14 ....................................................................................................................... 53 Figure 2.15 ....................................................................................................................... 54 Figure 2.16 ......................................................................................................................