Type V CRISPR-Cas effectors have revolutionized molecular diagnostics by facilitating the detection of nucleic acid biomarkers. However, their dependence on the presence of protospacer adjacent motif (PAM) sites on the target double-stranded DNA (dsDNA) greatly limits their flexibility as diagnostic tools. Here we present a novel method named PICNIC that solves the PAM problem for CRISPR-based diagnostics with just a simple ~10-min modification to contemporary CRISPR-detection protocols. Our method involves the separation of dsDNA into individual single-stranded DNA (ssDNA) strands through a high-temperature and high-pH treatment. We then detect the released ssDNA strands with diverse Cas12 enzymes in a PAM-free manner. We show the utility of PICNIC by successfully applying it for PAM-free detection with three different subtypes of the Cas12 family - Cas12a, Cas12b, and Cas12i. Notably, by combining PICNIC with a truncated 15-nucleotide spacer containing crRNA, we demonstrate PAM-independent detection of clinically important single-nucleotide polymorphisms (SNPs) with CRISPR. We apply this approach to detect the presence of a drug-resistant variant of HIV-1, specifically the K103N mutant, that lacks a PAM site in the vicinity of the mutation. Additionally, we successfully translate our approach to clinical samples by detecting and genotyping HCV-1a and HCV-1b variants with 100% specificity at a PAM-less site within the HCV genome. In summary, PICNIC is a simple yet groundbreaking method that enhances the flexibility and precision of CRISPR-Cas12-based diagnostics by eliminating the restriction of the PAM sequence.

P.K.J., S.R.R., E.K.V., G.M.S, and L.S.S are listed as inventors on a patent application related to the content of this work. P.K.J. is a co-founder of Genable Biosciences, Par Biosciences, and CRISPR, LLC. The remaining authors declare no competing interests.

This work was financially supported by funds from the UF, UF Herbert Wertheim College of Engineering, Shah Foundation Endowment Funds, NIH-NIAID R21AI156321, NIH-NIAID R21AI168795, and NIH-NIGMS R35GM147788. The funding sources did not have a role in the design of the study, the collection, analysis, or interpretation of data, nor in writing the manuscript.

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The IRB committee of the University of Florida gave ethical approval for this work under IRB202200294.

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