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Engineering High-Precision CRISPR-Cas9 Nuclease and Base Editor Technologies

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Engineering High-Precision CRISPR-Cas9 Nuclease and Base Editor Technologies Engineering High-Precision CRISPR-Cas9 Nuclease and Base Editor Technologies The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:40050081 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA "#$#%&'()*!+(*,-./#0(1(&)!2345.3-2617!861#!9:(;&/!<#0,)&%&*(#1! ! ! "!#$%%&'()($*+!,'&%&+(&#!-.! /)%*+!0&1'2&! (*! 31&!4&,)'(5&+(!*6!7*8&9:8)'!)+#!;&88:8)'!<$*8*=.! $+!,)'($)8!6:86$885&+(!*6!(1&!'&>:$'&5&+(%!6*'!(1&!#&='&&!*6! 4*9(*'!*6!?1$8*%*,1.! $+!(1&!%:-@&9(!*6! <$*91&5$%('.! ! ! ! A)'B)'#!C+$B&'%$(.! ;)5-'$#=&D!7)%%)91:%&((%! 7).!EFGH! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! I!EFGH!/)%*+!0&1'2&! "88!'$=1(%!'&%&'B&#J! ! Dissertation advisor: Dr. J. Keith Joung Author: Jason Gehrke Abstract The human genome, comprising approximately three billion nucleotides separated across 23 chromosomes, contains a vast amount of sequence space encoding instructions for the production of RNA and protein molecules that make up the functional components of the cell. Some changes in these genetic sequences produce phenotypic changes in organisms, such as genetically-heritable diseases in humans. Genome editing, loosely defined as creating targeted, sequence-specific changes in a desired cellular genome, promises to enable the correction of many disease-causing mutations. To enable these technologies to be used to their full potentials in research and therapeutic settings, it is necessary to engineer genome editing technologies with extreme specificity and fidelity to the desired target sequence compared to all other possible sequences in the target genome. This work focuses on engineering CRISPR-Cas9 RNA-guided nucleases and base editor technologies to enhance their precision and genome-wide specificities, thus enabling researchers to more effectively use these tools to study fundamental biological principles or to translate these technologies into clinical settings. Chapter 1 introduces relevant history of the genetic engineering field, including contemporary genome editing platforms such as CRISPR-Cas9 nuclease and base editor platforms. Chapter 2 describes efforts towards engineering CRISPR-Cas9 systems that are dependent on specific epigenetic contexts adjacent to their target site in order to successfully induce DNA double strand breaks, adding additional layers of complexity and regulation of activity to the CRISPR- Cas9 platform. iii Chapter 3 focuses on leveraging natural cytidine deaminase protein diversity, coupled with standard protein engineering techniques, to create base editor proteins able to programmably edit single nucleotides at desired on-target sites based on the sequence context of a given target base. The technology described in this chapter greatly enhances the ability of the base editor platform to correct many disease-causing genomic SNPs with fewer or no bystander nucleotide editing events at the on-target site, and greatly reduces the rates of off-target editing compared to the original base editor platform. Importantly, we demonstrate that this technology can be used to more efficiently and precisely correct a disease-causing mutation in a model cell line and in erythroid precursor cells derived from a patient bearing this mutation. iv Table of Contents Abstract ................................................................................................................................................. iii Table of Contents .................................................................................................................................... v Acknowledgements ............................................................................................................................... vii Chapter 1: Introduction ........................................................................................................................... 1 Repair of naturally-occurring or enzymatically-induced DNA lesions .................................................. 2 Overview of genome editing tools ....................................................................................................... 6 CRISPR and the democratization of genome editing .......................................................................... 10 Base editor technologies .................................................................................................................... 12 Genome-wide specificities of designer nucleases and base editors ..................................................... 14 Chapter 2: Engineering CRISPR-Cas9 for Improved Targeting Range, Delivery, and Control ............... 20 Engineering transcription factor-specific CRISPR-Cas9 nucleases ..................................................... 20 Engineering minimal Cas9 orthologs for double stranded DNA binding and nuclease activity ........... 29 Chapter 3: Engineering Sequence-Specific Cytidine Deaminases ........................................................... 32 Developing high-precision CRISPR-Cas9 base editors with minimized bystander and off-target mutations .......................................................................................................................................... 34 Supplementary Figures ...................................................................................................................... 51 Methods ............................................................................................................................................ 71 Chapter 4: Discussion and future directions ........................................................................................... 78 Supplementary Tables ........................................................................................................................... 84 Supplementary Table 1 ...................................................................................................................... 85 v Supplementary Table 2 ...................................................................................................................... 87 Supplementary Table 3 .....................................................................................................................102 Supplementary Table 4 .....................................................................................................................112 Supplementary Table 5 .....................................................................................................................116 Supplementary Table 6 .....................................................................................................................119 References ...........................................................................................................................................154! vi Acknowledgements I would like to express my sincere gratitude to Prof. J. Keith Joung for the years of scientific training and knowledge he has imparted on me, and the profound impact he has had on how I approach science, especially the field of genome editing. The creative autonomy given to me in proposing and pursuing ideas was essential for my development as a scientist. Though very few of my ideas became successful projects, each failed experiment taught me more about designing projects and experiments with scientific rigor and I couldn’t have asked for a more supportive mentor through this process. To my friends, particularly James Angstman, Diego Baptista, Jacques Carolan, Elliot Clark, Geoff Cockrell, Sandy Mattei, Grace Sager, Matt Smith and Cayla Zimmer: without you I would have never made it through graduate school. Your support and encouragement over the past five years, especially in difficult and trying times, has kept me going. To my family, and especially to my parents and sister: your support of my decisions, good or bad, has gotten me here, and I cannot thank you enough for all that you have done for me. Lastly, I’d like to thank all of my collaborators that I’ve worked with over the course of my graduate school experience. Whether we were successful in our endeavors or not, these were valuable experiences and I learned a great deal from every shared project. In particular, Prof. Dan Bauer and Drs. Yuxuan Wu, and Jing Zeng were instrumental in aiding the translation of the base editor technologies I developed to patient-derived cells. I also thank my advisory committee members, Profs. Mo Khalil, David Liu, and Alex Schier for the valuable advice and direction given to me over my graduate career, as well as the National Science Foundation for helping to fund my scientific education. vii Chapter 1: Introduction The genomes of organisms encode a set of instructions for the production of functional proteins and RNAs in the form of DNA sequence elements that collectively enable a given cell to perform standard housekeeping functions that meet the basic requirements of the cell, and also to sense external stimuli and respond through adaptation to their environment. Production of each of these elements is tightly regulated in the cell, and genomic alterations that result in misregulation of either protein-coding genes or non-coding RNA species can result in disease phenotypes in humans (Ptashne, 2014). The most well-understood
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