期刊
出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2113747118
关键词
CRISPR-Cas12a; genome editing; single-molecule FRET; DNA cleavage; metal ion
资金
- National Research Foundation of Korea - Ministry of Science and ICT of the Korean Government [2019R1C1C1008438, 2020M3A9I4038197, 2021R1A2C1007593]
- Gwangju Institute of Science and Technology (GIST) Research Institute
- National Research Foundation of Korea [2020M3A9I4038197, 2019R1C1C1008438, 2021R1A2C1007593] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
In the study, two conformational intermediates were identified in the process of Cas12a cleaving double-stranded DNA target, including PAM-distal DNA unwound conformation and the binding of the target strand to the catalytic site. These findings highlighted the importance of Mg2+ ions in promoting the binding and cleavage of the target strand, and proposed a Mg2+-dependent kinetic model for the mechanism of Cas12a achieving cleavage of the target DNA.
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 fluores-cence 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.
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