A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9.

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• The Cas9 endonuclease is a key tool in genome engineering, programmable via single-guide RNA (sgRNA).
• Improving the accuracy and efficiency of DNA targeting by Cas9 is an active area of research.
• The precise mechanism controlling Cas9's HNH nuclease domain activation at on-target DNA versus off-target sites is not fully understood.

Purpose of the Study:

• To elucidate the conformational dynamics of Streptococcus pyogenes Cas9 during DNA binding and cleavage.
• To identify the molecular checkpoint governing HNH nuclease domain activation.
• To understand how sequence mismatches influence Cas9 cleavage activity.

Main Methods:

• Single-molecule Förster resonance energy transfer (smFRET) was employed to monitor Cas9 conformational changes in real-time.
• Studies involved observing Cas9 interactions with DNA targets and guide RNAs.

Main Results:

• An intermediate conformational state of Cas9, acting as a checkpoint between DNA binding and cleavage, was identified.
• HNH domain docking into its active conformation requires divalent cations but not DNA strand scission.
• This active conformation is stable post-cleavage and sequence mismatches at off-target sites prevent the transition to the active state.

Conclusions:

• The identified checkpoint mechanism explains how Cas9 achieves selective DNA cleavage at on-target sites.
• Mismatches between sgRNA and off-target DNA prevent HNH domain activation, ensuring genome editing specificity.
• This work provides mechanistic insights into Cas9 accuracy, crucial for advancing genome engineering technologies.