Optimizing directed evolution of S. pyogenes Cas9

• Main Author: Lee, Shi Mun
• Other Authors: Tan Meng How
• Format: Theses and Dissertations
• Language: English
• Published: 2017
• Subjects: DRNTU::Engineering::Bioengineering
• Online Access: http://hdl.handle.net/10356/72364
• Institution: Nanyang Technological University

CRISPR/Cas9, since its discovery, has revolutionized the genome editing landscape; with one protein and a programmable RNA sequence, genome targeting has become achievable even in the non-experts’ hands. Despite its easy adaptation of the application, off-target and low efficiency have been issues to push its use in genetic therapy. In this research, attempts to enhance the efficiency of nuclease activity were attempted. Random mutagenesis has been performed in the region of the nuclease domain – RuvCII-HNH-RuvCIII. The enhanced mutants were expected to be isolated from a positive selection system where the survival of the host is linked to the Cas9 ability to severe the suicide plasmid. However, problems had arisen due to high background and toxicity issues; and attempts to optimize the condition to eliminate the problem has become the goal of this project.