Molecular mechanisms of <i>Streptococcus pyogenes</i> 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 used Streptococcus pyogenes (Sp) Cas9 revealed through these techniques.

A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets

“…Our results also imply that these three main factors affect SpCas9 activity in a similar way. Former studies have shown that both the mismatches and the fidelity increasing mutations in eSpCas9 and SpCas9-HF1 loosely trap SpCas9 in a catalytically inactive intermediate state and slow down the transition of the HNH domain to the active state 24 , 36 – 40 . Mismatches, both PAM-proximal and PAM-distal when bona fide off-targets are engaged, inhibit the HNH domain transition and tend to keep the nuclease in this inactive state 38 – 42 .…”