Advancing CRISPR-Cas Gene Editing Technologies: Engineering of Guide RNA, Donor Template, Editing Effector, and In Vivo Delivery

Abstract

Gene editing technologies have revolutionized various fields, from agriculture to medicine, by providing powerful tools to modify genetic materials. Early efforts, such as gene targeting, ZFN and TALEN, have laid the foundation for this field. In the past decade, CRISPR-Cas, derived from prokaryotic adaptive immune systems, has been re-engineered as gene editing tools, including nuclease editors, base editors, and prime editors. The simplicity, effectiveness, and versatility of these CRISPR-Cas gene editing tools have rapidly propelled their widespread use in both academia and industry. Despite the tremendous potential, many challenges arise during the development of CRISPR-Cas gene editing, and this thesis focuses on tackling some of the key ones. On one hand, I have dedicated my efforts to engineering gene editing components. This includes the synthesis of long guide RNA using click chemistry, enhancing the efficiency of homology-directed repair (HDR)-based editing using chemically modified donor templates, and improving modular prime editing platform by engineering effectors. On the other hand, I have also focused on the in vivo delivery of gene editors. Specifically, I have explored the first use of lipid nanoparticles for delivering chemically modified pegRNA and prime editor effector mRNA to achieve in vivo prime editing. Additionally, I have developed a fluorescence-based mouse reporter system to assess the in vivo performance of gene editors. Overall, the work presented in this thesis will greatly contribute to the advancement of CRISPR-Cas gene editing technologies, fostering progress in future research and therapeutic applications.