Molecular dynamics study on the mechanism of high-fidelity CRISPR-Cas9 system

• Main Author: Liu, Zhehui
• Other Authors: Mu Yuguang
• Format: Final Year Project
• Language: English
• Published: 2017
• Subjects: DRNTU::Science::Biological sciences::Biophysics
• Online Access: http://hdl.handle.net/10356/70480
• Institution: Nanyang Technological University

Evolving from adaptive immune system of bacteria, clustered regularly interspaced short palindromic repeat (CRISPR) - CRISPR associated (Cas9) system has been widely used as a tool for genome modification. Off-target effect is one of the major challenges of this system, but it has been largely reduced in a high-fidelity SpCas9 system (HF-SpCas9) in which four of the Cas9 residues (NRQQ) were mutated to alanine. However, the mechanism of how the mutated protein reduces the off-target effects remains obscure. To better understand this issue, molecular dynamics simulations of 7 SpCas9/gRNA:dsDNA systems were performed, including different mutations of protein residues and introducing mismatches in gRNA:dsDNA heteroduplex. Our simulation results suggested that HF-SpCas9 would have a more rigid recognition lobe and a more opened conformation between the catalytically active site and the target scissile phosphates. And the mutant systems also have demonstrated a weaker binding energy between Cas9 protein and gRNA:dsDNA complex. Hence, we concluded that the dynamic differences between WT and NRQQ mutant and the reduction in binding energy might be the key factors that would lower the off-target effects.