Architecture of a DNA-guided Cas12a

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• CRISPR/Cas systems are primarily known as RNA-guided nucleases.
• Understanding the mechanisms of DNA-guided RNA targeting is crucial for expanding CRISPR applications.

Purpose of the Study:

• To elucidate the structural basis of DNA-guided RNA recognition by Cas12a.
• To provide a structural framework for engineering Cas12a systems for novel applications.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structure of Acidaminococcus sp. Cas12a (AsCas12a).
• The structure was solved for AsCas12a complexed with a pseudo-DNA (ΨDNA) guide and an RNA target.

Main Results:

• The pseudo-DNA hairpin in the AsCas12a-ΨDNA-RNA complex bridges the recognition and nuclease lobes.
• This structure mimics a PAM-proximal duplex, positioning the spacer for canonical RNA-DNA heteroduplex formation.
• The REC lobe facilitates RNA-DNA heteroduplex formation, revealing the DNA-guided RNA recognition mechanism.

Conclusions:

• The determined structure provides a detailed framework for understanding AsCas12a's DNA-guided RNA targeting.
• This structural insight can guide the future engineering of Cas12a nucleases for diverse biotechnological applications.