Analytical chemistryOct 23, 2026

A CRISPR-Based Test Without PAM Requirements to Detect the katG Ser315Thr Mutation Linked to Isoniazid Resistance in Tuberculosis

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Oct 24, 2026

Abstract

The PddXNA-CRISPR platform demonstrated 90% concordance with sequencing results for detecting the katG Ser315Thr mutation in clinical samples.

Drug-resistance Tuberculosis (TB) poses a significant global health threat.
Current methods for detecting mutations associated with drug resistance face challenges in precision and simplicity.
The CRISPR-Cas12a system's effectiveness is limited by its requirement for a specific DNA motif for recognition.
An innovative XNA-CRISPR platform was developed to enable detection without this DNA motif, achieving single-base resolution.
Integration with droplet digital technology enhanced the sensitivity of the detection platform.
The approach provides a new tool for accurately identifying critical single-nucleotide variants in TB diagnostics.

Simplified

Drug-resistance Tuberculosis (TB) is a major threat to global public health. Simple and precise detection of the mutations associated with drug resistance is still a significant challenge. CRISPR-based nucleic acid detection has emerged as a transformative technology in molecular diagnostics due to its high specificity and programmability. However, the CRISPR-Cas12a system requires a protospacer adjacent motif (PAM) in the target double-stranded DNA (dsDNA) for efficient recognition and activation, which limits its application for detecting single-nucleotide variants (SNVs). To overcome this limitation, we leveraged high-affinity xenonucleic acids (XNAs) to engineer an XNA-CRISPR platform that combines a helicase and sequence-specific XNAs (such as peptide nucleic acid (PNA), locked nucleic acid (LNA), or 2'-modified RNA), to unwind the dsDNA, enabling them to invade and form stable hybrids, thereby displacing a single strand for PAM-independent Cas12a activation. This recognition mechanism, combining XNA hybridization and CRISPR verification, achieves single-base resolution. To bridge the sensitivity gap for clinical application, we integrated this platform with polydisperse droplet digital technology (PddXNA-CRISPR), which significantly enhanced the sensitivity. Clinical sputum samples were detected by using PddXNA-CRISPR for Mycobacterium tuberculosis (MTB) katG Ser315Thr mutation, which showed 90% concordance with sequencing results. This modular, amplification-free platform expands the CRISPR-based diagnostic toolkit and provides a versatile and sensitive approach for detecting critical SNVs.

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A CRISPR-Based Test Without PAM Requirements to Detect the katG Ser315Thr Mutation Linked to Isoniazid Resistance in Tuberculosis

Abstract

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