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]) and a secondary sgRNA-bound-like conformation (Cas9). Interestingly, increase in buffer KCl concentration led to a linear increase in the Cas9 population and a corresponding decrease in the Cas9 population. In contrast, changes in KCl concentration exerted the opposite effects on the Cas9 and Cas9 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.