Molecular mechanisms of Streptococcus pyogenes Cas9: a single-molecule perspective

Abstract

Cas9 is an RNA-guided endonuclease from the type II CRISPR-Cas system that employs RNA–DNA base pairing to target and cleave foreign DNA in bacteria. Due to its robust and programmable activity, Cas9 has been repurposed as a revolutionary technology for wide-ranging biological and medical applications. A comprehensive understanding of Cas9 mechanisms at the molecular level would aid in its better usage as a genome tool. Over the past few years, single-molecule techniques, such as fluorescence resonance energy transfer, DNA curtains, magnetic tweezers, and optical tweezers, have been extensively applied to characterize the detailed molecular mechanisms of Cas9 proteins. These techniques allow researchers to monitor molecular dynamics and conformational changes, probe essential DNA–protein interactions, detect intermediate states, and distinguish heterogeneity along the reaction pathway, thus providing enriched functional and mechanistic perspectives. This review outlines the single-molecule techniques that have been utilized for the investigation of Cas9 proteins and discusses insights into the mechanisms of the widely usedStreptococcus pyogenes (Sp) Cas9 revealed through these techniques.

Graphical abstract

Keywords

CRISPR / Cas9 / Single-molecule / Molecular mechanism / Nuclease / R-loop

Funding

This work was supported by the National Key Research and Development Program of China (2017YFA0106700), the National Natural Science Foundation of China (32022048 and 22104088), the Natural Science Foundation of Shanghai (19ZR1434100), and the China Postdoctoral Science Foundation (2021M692053). We sincerely apologize to authors whose work could not be included in this manuscript due to the space limitation.