Development of novel CRISPR-Cas9 protein scaffolds and base editors - engineering a smaller SpCas9

• Main Author: Teo, Danny Shun Xiang
• Other Authors: Tan Meng How
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2024
• Subjects: Medicine, Health and Life Sciences; Gene editing; CRISPR Cas9
• Online Access: https://hdl.handle.net/10356/181295
• Institution: Nanyang Technological University

CRISPR-Cas system holds immense potential for treatment of genetic diseases. However, therapeutic adoption of CRISPR-Cas9 system still faces issues with safety and efficient delivery in vivo. Gene editing with Cas9 can potentially treat genetic diseases at its root cause. However, this classically required Cas9 mediated DNA damage and subsequent repair by cellular DNA repair machinery. This led to several pitfalls – DNA damage and off target propensity of Cas9 raised safety concerns, while dependence on cellular homology directed repair machinery resulted in low efficiency. As such, alternate applications of Cas9 such as CRISPRa/i gene regulation and better gene editing technology such as base editing holds higher therapeutic potential in recent years. Another major weakness of Cas9 for therapeutic adoption is in vivo delivery. The Cas9 protein is large, which can be an issue for delivery of its genetic material or the protein itself. The current state-of-the-art for in vivo delivery of genetic material relies on recombinant adeno associated virus (rAAV), with FDA approved therapies such as Luxturna® and Zolgensma® as recent examples. However, the low packaging limit of rAAV precludes easy design and delivery, especially when considering alternative applications of Cas9 that often include additional fusion proteins on top of the already large Cas9 enzyme. This thesis explored rational truncation of SpCas9 and subsequent rational improvements to truncated Cas9 (tCas9). The functionality of tCas9 is demonstrated with CRISPRa/i gene regulation. To enable delivery with packaging limited rAAV, we then designed small, strong mammalian promoters that could enable robust expression of rAAV-delivered Cas9 constructs. Next, we demonstrated the compatibility of tCas9 with base editor fusions. We also showed that Cas9 is compatible with recently identified mutations that improved specificity and expanded PAM targetability, highlighting the value of tCas9 over existing smaller Cas9 orthologues. Finally, we explored in vivo delivery methods of tCas9 constructs, through both rAAV and circular RNA delivery modes.