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Anal Chem ; 95(23): 8879-8888, 2023 06 13.
Article in English | MEDLINE | ID: mdl-37252785

ABSTRACT

CRISPR/Cas12a has been believed to be powerful in molecular detection and diagnostics due to its amplified trans-cleavage feature. However, the activating specificity and multiple activation mechanisms of the Cas12a system are yet to be elucidated fully. Herein, a "synergistic activator effect" is discovered, which supports an activation mechanism that a synergistic incorporation of two short ssDNA activators can promote the trans-cleavage of CRISPR/Cas12a, while either of them is too short to work independently. As a proof-of-concept example, the synergistic activator-triggered CRISPR/Cas12a system has been successfully harnessed in the AND logic operation and the discrimination of single-nucleotide variants, requiring no signal conversion elements or other amplified enzymes. Moreover, a single-nucleotide specificity has been achieved for the detection of single-nucleotide variants by pre-introducing a synthetic mismatch between crRNA and the "helper" activator. The finding of "synergistic activator effect" not only provides deeper insight into CRISPR/Cas12a but also may facilitate its expanded application and power the exploration of the undiscovered properties of other CRISPR/Cas systems.


Subject(s)
Biosensing Techniques , CRISPR-Cas Systems , DNA, Single-Stranded , Nucleotides , RNA, Guide, CRISPR-Cas Systems
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