CRISPR-Based Diagnostics for Rapid Detection of Mycobacterium tuberculosis: Current Advances and Future Perspectives- A Systematic Review

Abstract

Background: Tuberculosis (TB) is one of the most common causes of mortality among infectious diseases around
the world. In 2022, the following figures were recorded: 10.6 million cases and 1.3 million deaths. Traditional
diagnoses such as smear microscopy, culture, and molecular diagnostic tests such as GeneXpert have serious flaws
in sensitivity, turnaround time, and suitability in limited resource settings.
Objective: The aim of the study is to assess the accuracy of diagnosis, the nature of operations, and the clinical benefit
of CRISPR-based assays to identify Mycobacterium tuberculosis (MTB) and drug-resistant strains.
Methods: The systematic literature search was performed in PubMed, ScienceDirect, Google Scholar, and Web of
Science using peer-reviewed articles regarding these categories that were published in the period between January
2015 and March 2025. Articles assessing the use of CRISPR-based diagnostics in the detection of the presence of
MTB in clinical samples were identified. The sensitivity, specificity, limit of detection (LOD), turnaround time, and
target gene data were extracted and synthesized. Quality was measured based on QUADAS-2.
Findings: 47 studies that fit the inclusion criteria were on hand, including 23 original research articles, 14 validation
studies, and 10 systematic reviews. CRISPR-based assays showed sensitivities of between 86.8 and 99.3 percent and
specificities of between 94 percent and a hundred percent using a variety of clinical samples. The detection limits
were between 0.5 and 15 copies/mL with a turnaround time of 60-90 minutes—much quicker than culture (4-8 weeks)
and identical to GeneXpert. Characteristic target sequences were IS6110 (in 94 percent of the strains of MTB),
IS1081, and 16S rRNA. A number of assays indicated the ability to concurrently detect mutations that were resistant
to drugs in rpoB (rifampicin), katG (isoniazid), and gyrA (fluoroquinolones). CRISPR systems such as SHERLOCK
(Cas13), DETECTR (Cas12a), and TB-QUICK (Cas12b) were found to be workable in sputum as well as
bronchoalveolar lavage and plasma-derived cell-free DNA. The combination of lateral flow strips and smartphone-
compatible readouts made the technology instrument-free and eligible to be deployed at the point of care.
Conclusion: CRISPR-based diagnostics are highly accurate and fast with a turnaround time and multiplexing
features, which meets WHO targets on TB detection. Nevertheless, heterogeneity of the study design, field validation,
and regulatory issues limit large-scale deployment at this time. It is justified that there should be further standardized
multicenter trials and that they should become part of national TB programs.