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

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 concentration led to a linear increase in the Cas9^X population and a corresponding decrease in the Cas9^apo population. In contrast, changes in KCl concentration exerted the opposite effects on the Cas9^X and Cas9^apo populations in the presence of sgRNA. Collectively, our findings by using KCl concentration as the probe suggest that Cas9 might employ a conformational sampling mechanism, in addition to the more common induced-fit mechanism established by us previously, for sgRNA binding.