AI Article Synopsis

  • The CRISPR/Cas12a system is effective for molecular diagnostics thanks to its precision in DNA recognition and cleavage, but previous regulation methods were complicated and costly.
  • Researchers introduced a novel method called FRAME, which uses flap endonuclease 1 (FEN1) to control the activity of Cas12a by regulating the PAM's accessibility in DNA.
  • The FRAME strategy not only enhances Cas12a's function but also allows for the detection of multiple targets using a single CRISPR RNA, showcasing its potential for improved medical diagnostics.

Article Abstract

CRISPR/Cas12a system, renowned for its precise recognition and efficient nucleic acid cleavage capabilities, has demonstrated remarkable performance in molecular diagnostics and biosensing. However, the reported Cas12a activity regulation methods often involved intricate CRISPR RNA (crRNA) structural adjustments or costly chemical modifications, which limited their applications. Here, we demonstrated a unique enzyme activity engineering strategy using flap endonuclease 1 (FEN1) to regulate the accessibility of the protospacer adjacent motif (PAM) module in the double-stranded DNA activator (FRAME). By identifying the three-base overlapping structure between the target inputs and substrate, FEN1 selectively cleaved and released the 5'-flap containing the 'TTTN' sequence, which triggered the secondary cleavage of FEN1 while forming a nicked PAM, ultimately achieving the sensitive switching of Cas12a's activity. The FRAME strategy exemplified the 'two birds with one stone' principle, as it not only precisely programmed Cas12a's activity but also simultaneously triggered isothermal cyclic amplification. Moreover, the FRAME strategy was applied to construct a sensing platform for detecting myeloperoxidase and miR-155, which demonstrated high sensitivity and specificity. Importantly, it proved its versatility in detecting multiple targets using a single crRNA without redesign. Collectively, the FRAME strategy opens up a novel avenue for modulating Cas12a's activity, promising immense potential in the realm of medical diagnostics.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11514456PMC
http://dx.doi.org/10.1093/nar/gkae804DOI Listing

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