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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• RNA-guided endonucleases, including CRISPR-Cas systems, are crucial for genome editing.
• CRISPR-Cas9, -Cas12, and related proteins utilize guide RNAs to target specific DNA sequences.
• The precise mechanisms of DNA recognition by these enzymes 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 target DNA interrogation by Cas12a.

Main Methods:

• Utilized structural and biochemical methods.
• Performed Cryo-electron microscopy (Cryo-EM) analysis of a Cas12a-RNA-DNA complex.
• Employed fluorescence-based conformational probing.

Main Results:

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

Conclusions:

• RNA-mediated DNA engineering by CRISPR-Cas proteins initiates with local DNA helix distortion.
• Cas12a's mechanism involves transient protein binding and helix destabilization for DNA recognition.
• Understanding these initial steps is vital for advancing CRISPR-based genome editing technologies.