Structure of the type V-C CRISPR-Cas effector enzyme.

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RNA-guided CRISPR-Cas nucleases are essential genome-engineering tools.
• Type V Cas12 family enzymes exhibit functional diversity.
• Cas12c2 utilizes CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) for DNA targeting with a TN PAM.

Purpose of the Study:

• To elucidate the structural basis of Cas12c2 function.
• To understand the mechanism of guide RNA binding and DNA recognition.
• To investigate the unique RNA processing activity of Cas12c2.

Main Methods:

• Cryo-electron microscopy (cryo-EM) for structural determination.
• Biochemical assays to analyze enzyme activity and interactions.

Main Results:

• Determined cryo-EM structures of Cas12c2-guide RNA and Cas12c2-guide RNA-target DNA complexes.
• Revealed an unexpected X-junction architecture formed by crRNA and tracrRNA.
• Identified specific hydrogen-bonding interactions between Cas12c2 and the TN PAM, recognizing a single T nucleotide.
• Demonstrated a unique crRNA processing mechanism mediated by the RuvC catalytic site.

Conclusions:

• The findings provide detailed structural insights into the Cas12c2 CRISPR-Cas effector.
• This work enhances the mechanistic understanding of diverse type V CRISPR-Cas systems.
• The unique features of Cas12c2 may offer new possibilities for genome engineering applications.