CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• RNA-guided endonucleases, including CRISPR-Cas systems, are crucial for genome editing.
• The precise mechanisms of DNA target recognition by CRISPR-Cas proteins are not fully elucidated.

Purpose of the Study:

• To investigate the early steps of DNA recognition by Cas12a protein-guide RNA complexes.
• To elucidate the structural and biochemical basis of Cas12a-mediated DNA target engagement.

Main Methods:

• Cryo-electron microscopy (Cryo-EM) structural analysis of Cas12a-RNA-DNA complexes.
• Biochemical assays and fluorescence-based conformational probing.

Main Results:

• Cas12a induces DNA bending and transient nucleotide flipping to facilitate DNA-RNA hybridization.
• Cas12a destabilizes the DNA helix, promoting target discovery and engagement.
• The DNA interrogation mechanism shares similarities with CRISPR-Cas9, despite evolutionary differences.

Conclusions:

• RNA-mediated DNA interference by CRISPR-Cas proteins initiates with local DNA helix distortion.
• Cas12a employs a DNA bending and nucleotide flipping strategy for target recognition.