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bioRxiv · 10.1101/2025.06.27.661965

One-Pot Isothermal Linear Amplification and Cas12a-based Nucleic Acid Detection

Sinan, S.·Kooistra, R.·Rajaraman, K.·Islam, Z.·Madan, D.·Nalefski, E.·Finkelstein, I. J.
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Abstract

CRISPR-based nucleic acid diagnostics are a promising class of point-of-care tools that could dramatically improve healthcare outcomes for millions worldwide. However, these diagnostics require nucleic acid pre-amplification, an additional step that complicates deployment to low resource settings. Here, we developed CATNAP (Cas trans-nuclease detection of amplified products), a method that integrates isothermal linear DNA amplification with Cas12a detection in a single reaction. CATNAP uses a nicking enzyme and DNA polymerase to continuously generate single-stranded DNA, activating Cas12as trans-cleavage activity without damaging the template. We optimized enzyme combinations, buffer conditions, and target selection to achieve high catalytic efficiency. CATNAP successfully distinguished between high- and low-risk HPV strains and detects HPV-16 in a cervical cancer crude cell lysate at room temperature with minimal equipment, offering advantages over PCR-based approaches. We conclude that CATNAP bridges the sensitivity gap in CRISPR diagnostics while maintaining simplicity, making accurate disease detection more accessible in resource-limited settings.

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Sinan, S., Kooistra, R., Rajaraman, K., Islam, Z., Madan, D., Nalefski, E., Finkelstein, I. J.. 2025-06-29. One-Pot Isothermal Linear Amplification and Cas12a-based Nucleic Acid Detection. https://doi.org/10.1101/2025.06.27.661965

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