Journal: Nucleic Acids Research

Article Title: crRNA scaffold remodeling controls CRISPR–Cas12a activity for enhanced performance

doi: 10.1093/nar/gkag167

Figure Lengend Snippet: DNA blocker mediated crRNA scaffold remodeling regulates Cas12a trans - and cis- cleavage activities. ( A ) Schematic of length-dependent scaffold occlusion at the 5′ end for regulating Cas12a cis- and trans-cleavage activities. ( B and C ) Trans -cleavage fluorescence signals (bar plot) and cleavage efficiencies (line plot with markers) after 1 h for blockers introduced from the 5′ end (B) or 3′ end (C) of the scaffold. ( D and E ) Cis -cleavage fluorescence signals (bar plot) and cleavage efficiencies (line plot with markers) after 1 h for blockers introduced from the 5′ end (D) or 3′ end (E) of the scaffold. ( F ) Domain structure of LbCas12a (PDB 5ID6). ( G ) Molecular docking simulation of LbCas12a with conventional crRNA and ssDNA activator. ( H ) Molecular docking simulation of LbCas12a with scaffold-closed crRNA and ssDNA activator. Each experiment was repeated three times.

Article Snippet: All reactions were assembled in a 20 μl volume containing the crRNA/blocker duplex (50 nM/75 nM), LbCas12a (50 nM), and NEBuffer r2.1 (1×), with TE Mg 2+ buffer (12.5 mM) added to reach the final volume.

Techniques: Fluorescence