# Identification of thermotolerant non-canonical PAMs for robust one-pot CRISPR-Cas12a detection

## TL;DR

Researchers developed a new CRISPR-based detection system that uses non-canonical PAMs at higher temperatures for faster and more accurate pathogen detection.

## Contribution

The study identifies thermotolerant non-canonical PAMs that enable improved CRISPR-Cas12a detection performance at elevated temperatures.

## Key findings

- Non-canonical PAMs show trans-cleavage activity comparable to canonical PAMs at 45 °C or higher.
- Combining non-canonical PAMs with elevated temperatures improves sequence discrimination and detection performance.
- POP-CRISPR detects pathogens like HPV and Mycoplasma pneumoniae rapidly and accurately in clinical samples.

## Abstract

The canonical PAM site TTTV (where V = A, G, or C) is widely used in the design of CRISPR-Cas12a systems for both genome editing and diagnostic applications. Although several non-canonical protospacer-adjacent motifs (PAM) have been identified, they generally exhibit weak Cas12a cleavage activity. In this study, we find that increasing the reaction temperature to 45 °C or higher allows the identification of numerous non-canonical PAMs with trans-cleavage activity comparable to that of canonical PAMs, while displaying only weak cis-cleavage activity. Moreover, we observe that combining these non-canonical PAMs with elevated temperatures significantly enhances the Cas12a system’s ability to discriminate highly similar sequences. Based on these findings, we develop a non-canonical PAM-mediated, poikilothermal, one-pot CRISPR-Cas12a detection platform (POP-CRISPR), which demonstrates substantial improvements in sensitivity, specificity, speed, and target adaptability for nucleic acid detection compared to existing methods. These advantages are validated through the reliable detection of clinical samples, including those of Human papillomavirus (HPV), Mycoplasma pneumoniae (MP), and its drug-resistant strains. Additionally, we show that POP-CRISPR enables rapid, on-site pathogen detection within 20 min, using a fast sample processing protocol and a miniaturized detection device.

CRISPR-Cas12a diagnostics are limited by strict PAM requirements. Here, authors show that higher temperatures activate numerous noncanonical PAMs, enabling a versatile one-pot platform that improves sensitivity, specificity, and rapid on-site pathogen detection.