# A One-Pot CRISPR/Cas12a-Based Platform for Contamination-Free Nucleic Acid Amplification Detection

## TL;DR

This paper introduces a contamination-free CRISPR-based method for detecting nucleic acids in a single reaction step, reducing the risk of cross-contamination.

## Contribution

A one-pot CRISPR/Cas12a system that prevents contamination by incorporating PAM sites into primers and using pre-stored detection complexes.

## Key findings

- The method effectively degrades up to 10^6 copies of carryover contaminants within one hour.
- The system enables endpoint detection without open-tube manipulation, reducing contamination risks.
- Incorporating PAM sites into primers allows selective cleavage of contaminants while preserving target DNA.

## Abstract

CRISPR-Cas12a enables rapid and specific detection of PCR/LAMP (loop-mediated isothermal amplification) reaction products; however, this approach often requires open-tube manipulation, rendering it prone to cross-contamination. Here, we developed a novel one-pot reaction system that eliminated carryover contamination and facilitated endpoint detection using a CRISPR/Cas12a-based system. We leveraged the dependence of the CRISPR-Cas12a cleavage system on the protospacer-adjacent motif (PAM) to design PCR/LAMP primers that incorporated the PAM site (TTT) into amplified DNA. Pre-incubation of Cas12a with crRNA1 and crRNA2 using PCR/LAMP resulted in efficient cleavage of cross-contaminating DNA, while the target gene remained intact due to the lack of PAM sites. Furthermore, a Cas12a-detection complex (comprising Cas12a, crRNA3, trehalose, and the ssDNA probe) pre-stored on the lid was introduced to mix with the PCR/LAMP amplicons, which triggered the non-specific cleavage of fluorescent probes for direct visual detection under a blue LED instrument. This method effectively degraded up to 106 copies of carryover contaminants within one hour, demonstrating the potential of one-pot detection methods in complex samples.