Structural and mechanistic insights into the sequential dsDNA cleavage by SpCas12f1

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• Miniature CRISPR-Cas12f1 effector complexes are valuable for genome engineering.
• Cas12f1 functions as a homodimer using a single guide RNA.
• The precise reaction dynamics of Cas12f1 remained largely uncharacterized.

Purpose of the Study:

• To investigate the DNA recognition and cleavage mechanisms of Syntrophomonas palmitatica Cas12f1 (SpCas12f1).
• To elucidate the dynamic events during SpCas12f1-mediated DNA processing.

Main Methods:

• X-ray crystallography to determine SpCas12f1-DNA complex structure.
• Single-molecule magnetic tweezers for dynamic measurements.
• Ensemble kinetic assays for reaction rates.

Main Results:

• SpCas12f1 forms stable 18 bp R-loops, stabilized by local protein-R-loop interactions.
• DNA cleavage is mediated by a single catalytic center.
• Nontarget strand degradation occurs rapidly (∼11 bp) at random sites.
• Target strand cleavage is a slower process contingent on nontarget strand nicking.

Conclusions:

• SpCas12f1 employs a unique mechanism involving R-loop stabilization for DNA targeting.
• The sequential cleavage of nontarget and target strands dictates the overall SpCas12f1 activity.
• Understanding these dynamics is crucial for optimizing SpCas12f1 in genome engineering.