Breaking the PAM Restriction: A Universal Double Stranded DNA Detection Method Based on the Sticky End-Mediated CRISPR/Cas12a Coupled RPA and Its Application to KRAS G12C Single Base Mutations

Nov 11, 2025Analytical chemistry

A Universal Method for Detecting Double-Stranded DNA Using CRISPR and DNA Amplification Applied to KRAS G12C Single Base Mutations

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Abstract

A detection limit of 40 aM was achieved for a universal PAM-free CRISPR/Cas12a system.

The developed method integrates sticky end-mediated CRISPR/Cas12a with recombinase polymerase amplification (RPA).
Incorporating NlaIII recognition sites into RPA primers allows for precise cleavage of amplification products, generating uniform sticky ends.
Utilizing sticky end dsDNA significantly enhances Cas12a activity compared to flat end dsDNA with PAM sites.
The system successfully identifies KRAS G12C mutations at a frequency of 0.1%, aligning with genomic DNA results from FastNGS.
Preliminary exploration of a one-tube detection strategy effectively streamlined the operational process and reduced aerosol contamination.

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The CRISPR/Cas12a system facilitates efficient and specific nucleic acid detection, but its dependence on Protospacer Adjacent Motif (PAM) sequences and the complexity of existing sticky end-based methods pose challenges for stable and portable applications. To address these issues, this study developed a universal dsDNA detection method by integrating the sticky end-mediated CRISPR/Cas12a with recombinase polymerase amplification (RPA). By incorporating NlaIII recognition sites into RPA primers, precise cleavage of amplification products was achieved, generating uniform sticky ends and eliminating reliance on PAM sites. In comparison to flat end dsDNA containing PAM sites, the use of sticky end dsDNA significantly enhanced Cas12a activity. This strategy demonstrated sensitivity and specificity, achieving a detection limit of 40 aM and successfully identifying KRAS G12C mutations at a frequency of 0.1%, with genomic DNA results aligning with those obtained from FastNGS. Furthermore, we preliminarily explored a one-tube detection strategy, which effectively streamlined the operational process and reduced aerosol contamination. In summary, we established a simple, sensitive, and universal PAM-free CRISPR/Cas12a detection platform that integrates the advantages of isothermal amplification with a standardized sticky end design, thereby offering broad application prospects in molecular diagnostics and clinical translation.