Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but th...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2021-12, Vol.118 (49), p.1-8
Main Authors: Son, Heyjin, Park, Jaeil, Hwang, Injoo, Jung, Youngri, Bae, Sangsu, Lee, Sanghwa
Format: Article
Language:English
Subjects:
Binding
Biological Sciences
Cleavage
Conformation
CRISPR
Deoxyribonucleic acid
DNA
DNA damage
Endonuclease
Energy transfer
Fluorescence
Fluorescence resonance energy transfer
Genomes
Intermediates
Magnesium
Nucleic acids
Unwinding
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Summary:CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)–distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.2113747118