Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen‐Deuterium Exchange Mass Spectrometry

Polina V. Zhdanova, Alexander A. Chernonosov, Daria V. Prokhorova, Grigory A. Stepanov, Lyubov Yu Kanazhevskaya, Vladimir V. Koval

Результат исследования: Научные публикации в периодических изданияхстатьярецензирование

Аннотация

The Cas9 endonuclease is an essential component of the CRISPR–Cas‐based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR–Cas9 system. A deep under-standing of the Cas9 mechanism and its structural‐functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX‐MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to un-dergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX‐MS for the elucidation of mechanistic aspects of Cas9 functioning.

Язык оригиналаанглийский
Номер статьи1129
ЖурналInternational Journal of Molecular Sciences
Том23
Номер выпуска3
DOI
СостояниеОпубликовано - 1 февр. 2022

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