Expanding the Target Range of a Type IIB Cas9 Enzyme Through Evolution-Guided Engineering

Abstract

The discovery and development of CRISPR-Cas9 has revolutionized genome edit ing and enabled the development of novel therapeutic approaches. However, many disease-causing mutations are still inaccessible due to the absence of relevant PAM sequences within the therapeutic editing window. This master’s thesis employed di rected evolution to generate ePsCas9 variants with expanded PAM recognition capa bilities, enabling the targeting of disease-causing mutations previously inaccessible. Different library generation approaches and selection strategies were systematically explored. Selected variants obtained from the directed evolution were screened using a luciferase reporter system and four lead candidates (pCN1, pCN12, pCN15, and pCN27) were identified. A novel PAM-determination assay was established to profile motif recognition, revealing that pCN1, pCN12, and pCN15 exhibit relaxed recog nition of NRG or NDG motifs. This PAM assay provides a valuable tool for char acterizing Cas9 variants and was successfully applied to determine the NNNGAA recognition motif of another novel proprietary AstraZeneca Cas9 enzyme. Genome editing analysis demonstrated that pCN12 and pCN15 achieved equal or greater activity than the established SpRY variant at two of three NAG-PAM target se quences, while pCN27 showed enhanced NGC-PAM activity compared to wild-type ePsCas9.