One-Pot CRISPR-Cas12a Assay for Rapid Pulmonary Tuberculosis Diagnosis from Sputum: A Review

Abstract

Introduction- Pulmonary tuberculosis (TB) remains a major global health challenge, with persistent diagnostic gaps despite the scale-up of nucleic acid amplification tests such as CBNAAT (Xpert MTB/RIF) and Truenat MTB-RIF. These platforms, while highly sensitive and WHO-endorsed, are limited in many high-burden settings by cost, infrastructure requirements, and dependence on stable power and cartridge supply chains. One-pot CRISPR-Cas12a–based assays have emerged as a promising alternative, integrating isothermal amplification and CRISPR-mediated detection in a single closed tube, enabling rapid, highly specific identification of Mycobacterium tuberculosis and rifampicin resistance directly from sputum, with sample-to-result times typically under 60–80 minutes. This review summarizes the principles, assay designs, analytical performance, and readout formats (fluorescent, colorimetric, and lateral flow) of one-pot CRISPR-Cas12a diagnostics and compares their sensitivity, specificity, turnaround time, operational feasibility, and biosafety profile with CBNAAT and Truenat. We also discuss current technical and clinical challenges, including sample preparation, inhibitors, HIV co-infection, and field implementation in resource-limited settings. Finally, we highlight regulatory and quality considerations, potential diagnostic algorithms, and future directions such as multiplexing, amplification-free formats, and scalable surveillance applications. Together, the evidence suggests that one-pot CRISPR-Cas12a assays could complement, and in some contexts partially substitute, existing molecular platforms for decentralized, rapid TB diagnosis.

Objectives: Tuberculosis (TB) is among the top ten infectious disease threats globally and ranks as the leading cause of death from a single infectious agent. An estimated 10.6 million new TB cases occurred in 2021, with approximately 1.6 million deaths, according to the World Health Organization (WHO) Global Tuberculosis Report 2022. Early diagnosis and appropriate treatment are essential for successful TB control. However, existing diagnostic methods have one or more of the following limitations: delays of weeks to months in result delivery; insensitivity to the Mycobacterium tuberculosis (Mtb) pathogen; inability to detect active disease; inability to identify cases with low bacterial load, extreme drug resistance, or smear-negative sputum; reliance on clinical facilities far removed from the patient; a requirement for skilled personnel or specialized equipment; high cost; or unsuitability for human specimens or nonconventional sample matrices ¹. Such limitations result in missed and misdiagnosed cases, even in countries with high-level systems. Therefore, diagnostic testing is a global priority in the effort to eradicate TB¹².

The laboratory-scale CRISPR-Cas system—one of the most promising technologies for highly sensitive nucleic acid detection—has demonstrated substantial diagnostic potential in automated or manual laboratory workflows. The CRISPR-Cas12 (C2c2) variant is particularly suitable because signal readout occurs via cleavage of a fluorophore-quencher-labeled single-stranded DNA probe rather than the template strand. The one-pot CRISPR-Cas12a assay that combines nucleic-acid amplification and nucleic-acid interrogation into a single diagnostic step for the first time has been adapted for detection of the Mtb pathogen from sputum samples within 80 minutes, when coupled with cross-priming amplification.¹

MethodsThe diagnosis of tuberculosis (TB) primarily relies on microbiological methods that identify Mycobacterium tuberculosis in patient samples. These are time-consuming and are often limited to well-equipped laboratories ¹. CRISPR diagnostic platforms, integrated with nucleic acid amplification reactions, offer the needed sensitivity while requiring significantly less and simpler instrumentation. However, the multi-step processing increases the risk of sample contamination and complicates operational logistics.

One-Pot assays that couple amplification and CRISPR-enabled detection in a single reaction have shown great potential. In these formats, the entire reaction kit can be pre-mixed and stored in low-cost bulk, and the sample to-answer time is further shortened. M. tuberculosis-specific amplification targets that allow the straightforward construction of one-pot assays have been established. These amplification guides prompted the development of the One-Pot Tuberculosis (TB) assay combining CRISPR-Cas12a and Reverse-Transcription-Polymerase-Chain-Reaction (RT-PCR) in a single tube. Standard-free RNA extraction can be performed using 3% sodium dodecyl-sulfate and the system can detect nascent TB RNA 50 min after sampling. The whole assay time, including sample extraction and detection, is below 80 min, demonstrating a strong potential to serve as a new point-of-care diagnostic. ¹³

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Conclusions: The current landscape of TB diagnostics is evolving to meet the need for rapid tests that provide timely results to inform treatment initiation. One-Pot CRISPR-Cas12a assays based on the detection of M. tuberculosis in sputum, deployed during the TB-CAMP visit, demonstrate significant advancements over existing methods. Together with assured performance in the presence of HIV co-infection and consideration of implementation challenges, these assays offer opportunities to mitigate the devastating impact of TB and support global elimination efforts.¹