Development of One-pot LAMP CRISPR Diagnostics for Infectious Disease Detection

Abstract

The CRISPR/Cas system has emerged as a promising nucleic acid diagnostic tool beyond genome editing due to its high sequence specificity. Since pathogen infections can lead to community transmission and public health crises, rapid and accurate detection of pathogen-derived nucleic acids is essential. One-pot CRISPR diagnostic systems, which integrate isothermal amplification and CRISPR-based detection in a single tube, have therefore attracted attention as next-generation platforms for infectious disease diagnosis. In particular, LAMP enables rapid amplification of low-abundance target nucleic acids, while CRISPR/Cas systems provide sequence- specific target recognition. However, premature activation of Cas-mediated cis-cleavage before sufficient amplification can interfere with the amplification reaction and reduce detection sensitivity. Thus, a temperature- adaptable cis-cleavage control strategy is required, especially for high-temperature LAMP-based one-pot systems. In this study, Locked Nucleic Acid （LNA） was introduced into a crRNA-complementary regulator to control Cas cis-cleavage activity under high temperature conditions. By adjusting the number of LNA substitutions, Cas activity could be precisely regulated near 60°C without altering the crRNA spacer sequence, enabling stable one-pot integration of LAMP amplification and CRISPR detection. This LNA-based strategy improved reaction efficiency under high-temperature conditions and offers scalability for diverse pathogen targets without target- dependent spacer redesign. Validation using 60 patient-derived clinical samples demonstrated sensitivity and specificity comparable to qPCR. Overall, this study presents an LNA-based cis-cleavage control strategy for constructing a modular and adaptable One-pot LAMP CRISPR diagnostics for diverse infectious pathogen detection.