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 an...

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Bibliographic Details
Published in:Chemical communications (Cambridge, England) England), 2024-11, Vol.6 (89), p.1387-139
Main Authors: Son, Heyjin, Kang, Youngjae, Song, Yo Han, Park, Jaeil, Lee, Sanghwa
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
Catalytic Domain
Cleavage
CRISPR-Associated Proteins - chemistry
CRISPR-Associated Proteins - metabolism
CRISPR-Cas Systems
DNA - chemistry
DNA - metabolism
Endodeoxyribonucleases - chemistry
Endodeoxyribonucleases - metabolism
Fluorescence Resonance Energy Transfer
Steric hindrance
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Summary: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.
ISSN:1359-7345
1364-548X
1364-548X
DOI:10.1039/d4cc04716h