Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR-Cas12f nuclease.

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• CRISPR-Cas systems are essential tools for genome editing.
• Type V CRISPR-Cas nucleases exhibit diverse mechanisms and sizes.
• Cas12f (Cas14) is a notably small nuclease with potential for novel applications.

Purpose of the Study:

• To elucidate the structural basis of substrate recognition and DNA cleavage by the Cas12f nuclease.
• To investigate the activation mechanism of the asymmetric Cas12f dimer.
• To explore the activity of truncated guide RNAs with Cas12f.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structures of Cas12f complexes.
• Structural analysis of Cas12f bound to sgRNA and target DNA.
• Biochemical assays to assess the activity of truncated sgRNAs.

Main Results:

• Determined cryo-EM structures of Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes.
• Revealed an asymmetric Cas12f dimer utilizing one sgRNA for T-rich PAM dsDNA cleavage.
• Identified a conserved activation mechanism involving conformational changes and RuvC domain activation.
• Demonstrated that truncated sgRNAs are active for DNA cleavage.

Conclusions:

• Cas12f employs a unique asymmetric dimeric structure and conserved activation mechanism for DNA targeting.
• The small size and activity of Cas12f, even with truncated RNAs, offer significant potential for genome editing technologies.
• Structural insights facilitate the development of novel CRISPR-Cas12f-based genome editing tools.