Conformational transitions of Streptococcus pyogenes Cas9 induced by salt and single-guide RNA binding.

ABSTRACT: Streptococcus pyogenes (Sp) Cas9 has been widely utilized to edit genomes across diverse species. To achieve high efficiency and specificity as a gene-editing enzyme, Sp Cas9 undergoes a series of sequential conformational changes during substrate binding and catalysis. Here, we employed single-molecule FRET techniques to investigate the effect of different KCl concentrations on conformational dynamics of Sp Cas9 in the presence or the absence of a single-guide RNA (sgRNA). In the absence of sgRNA and at low KCl concentrations (75 mM), apo Cas9 surprisingly exhibited two distinct conformations: a primary autoinhibited open conformation (apo Cas9 conformation [Cas9^apo]) and a secondary sgRNA-bound-like conformation (Cas9^X). Interestingly, increase in buffer KCl concent

SUBMITTER: He Y 

PROVIDER: S-EPMC11791316 | biostudies-literature | 2025 Feb

REPOSITORIES: biostudies-literature