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Updated: Jun 4, 2025
A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
Published on: September 2, 2021
Conformational transitions of Streptococcus pyogenes Cas9 induced by salt and single-guide RNA binding
Yufan He1, Nikita Zalenski1, Anthony A Stephenson2
1Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida, USA.
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 [Cas9apo]) and a secondary sgRNA-bound-like conformation (Cas9X). Interestingly, increase in buffer KCl concentration led to a linear increase in the Cas9X population and a corresponding decrease in the Cas9apo population. In contrast, changes in KCl concentration exerted the opposite effects on the Cas9X and Cas9apo 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.
Insights
The study reveals that Streptococcus pyogenes Cas9 protein exists in distinct conformations, influenced by salt concentration and guide RNA presence. This suggests Cas9 utilizes conformational sampling for guide RNA binding.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Streptococcus pyogenes Cas9 (SpCas9) is a key enzyme for genome editing.
- SpCas9 requires specific conformational changes for efficient DNA targeting and cleavage.
- Understanding these dynamics is crucial for optimizing gene-editing technologies.
Purpose of the Study:
- To investigate the conformational dynamics of SpCas9.
- To determine the influence of potassium chloride (KCl) concentration on SpCas9 conformations.
- To explore the role of KCl in SpCas9-sgRNA interactions.
Main Methods:
- Single-molecule Förster Resonance Energy Transfer (smFRET) techniques were employed.
- Conformational states of SpCas9 were analyzed at varying KCl concentrations.
- Experiments were conducted with and without single-guide RNA (sgRNA).
Main Results:
- In the absence of sgRNA, apo Cas9 adopted two conformations: an autoinhibited open state (Cas9apo) and an sgRNA-bound-like state (Cas9X).
- Increasing KCl concentration favored the Cas9X conformation while decreasing the Cas9apo population.
- Conversely, KCl affected the Cas9X and Cas9apo populations oppositely in the presence of sgRNA.
Conclusions:
- KCl concentration acts as a probe for SpCas9 conformational dynamics.
- Findings suggest SpCas9 may utilize a conformational sampling mechanism for sgRNA binding.
- This complements the previously established induced-fit model for SpCas9-sgRNA interaction.
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