Nuc domain electrostatics drive the trans cleavage activity of CRISPR-Cas12a.

ABSTRACT: The trans cleavage activity of Cas12a has been extensively used for the detection of biomolecules. Different Cas12a orthologues exhibit faster or slower trans cleavage kinetics, making some orthologues more suited for sensitive molecular detection. Ionic strength of reaction buffers and mutations that change the electrostatic environment near the RuvC active site have also been reported to strongly influence trans cleavage kinetics. Studying three commonly used Cas12a orthologues (FnCas12a, AsCas12a, and LbCas12a), we report that electrostatic interactions near the RuvC active site are critical for their trans cleavage activity. Alanine substitution of arginine and lysine residues in the Nuc domain can abolish trans cleavage while modestly reducing cis cleavage. Substitutions in the RuvC l

SUBMITTER: Newman A 

PROVIDER: S-EPMC12784958 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature