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Zhang Qian, Chen Ziting, Sun Bo. Molecular mechanisms of Streptococcus pyogenes Cas9: a single-molecule perspective[J]. Biophysics Reports, 2021, 7(6): 475-489. doi: 10.52601/bpr.2021.210021
Citation: Zhang Qian, Chen Ziting, Sun Bo. Molecular mechanisms of Streptococcus pyogenes Cas9: a single-molecule perspective[J]. Biophysics Reports, 2021, 7(6): 475-489. doi: 10.52601/bpr.2021.210021

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

doi: 10.52601/bpr.2021.210021
Funds:  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.
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  • Corresponding author: sunbo@shanghaitech.edu.cn (B. Sun)
  • Received Date: 25 June 2021
  • Accepted Date: 11 November 2021
  • Available Online: 24 February 2022
  • Publish Date: 31 December 2021
  • 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.
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