Effects of steric hindrance from single-stranded overhangs on target-strand loading into the Cas12a active site

CRISPR-Cas12a, an RNA-guided DNA endonuclease, induces double-strand breaks by cleaving the non-target strand (NTS) first, followed by the target strand (TS). Using single-molecule FRET with alternating-laser excitation, we found that steric hindrance from the 3′ overhangs of both the cleaved NTS and crRNA impedes TS loading into the catalytic core. Our study highlights the direct involvement of both 3′ NTS and crRNA overhangs in TS cleavage, offering insights into regulatory strategies for Cas12a cleavage reactions. Single-molecule FRET data reveals that the crRNA and cleaved NTS overhangs sterically inhibit TS loading, thereby reducing TS cleavage efficiency.