CRISPR-Cas12a: As a diagnostic tool for sensitive and specific detection of hotspot Mpox virus infection

The rapid and accurate detection of monkeypox virus (MPXV) is urgently needed to control outbreaks and limit its spread, as the virus continues to pose significant public health threats across Africa and globally. Traditional diagnostic methods often face challenges related to sensitivity, specificity, and time consumption, driving the demand for more advanced molecular diagnostic tools. CRISPR-Cas12a has emerged as a promising platform for MPXV diagnosis due to its exceptional sensitivity, specificity, and rapid turnaround time. Unlike conventional techniques, CRISPR-Cas12a uses RNA-guided DNA cleavage and trans-cleavage activities to detect viral DNA sequences with high precision, significantly reducing false positives by targeting conserved viral genes. The Cas12a-based detection of MPXV works via the trans-cleavage mechanism whereby the Cas12a enzyme. This trans-cleavage activity generates detectable signals indicating the presence of MPXV DNA. Cas12a detection systems can achieve high sensitivity with a limit of detection down to 1–10 viral DNA copies within about 30–40 min. The assays demonstrate excellent specificity in distinguishing MPXV from other orthopoxviruses, making them suitable for rapid point-of-care (POC) testing in resource-limited settings. Moreover, technological innovations integrating CRISPR-Cas12a with isothermal amplification methods like recombinase polymerase amplification (RPA) and the development of fluorescent and lateral flow strip-based assays have enhanced detection speed and adaptability to low-resource settings, enabling POC. Additionally, advances targeting amplification-free detection and the use of ultrasensitive biosensing platforms further simplify assay workflows while maintaining accuracy comparable to qPCR. Despite these advantages, challenges remain, especially regarding off-target effects, sample preparation optimization, and delivery mechanisms in complex clinical samples. Future research is essential to validate and refine CRISPR-Cas12a diagnostics for routine clinical use, improving scalability and regulatory compliance. In conclusion, CRISPR-Cas12a offers a transformative approach for rapid, sensitive, and specific MPXV detection, potentially revolutionizing outbreak surveillance and patient management. Continued interdisciplinary efforts are critical to overcoming present limitations and fully harnessing the technology's potential in combating MPXV infection. • CRISPR-Cas12a: Next-gen tool for rapid, accurate Mpox detection. • High sensitivity & specificity for hotspot Mpox virus infection. • RPA boosts CRISPR-Cas12a amplified Mpox detection. • Fluorescent/lateral flow assays for non-amplified MPXV detection. • Functional testing validates CRISPR-Cas12a's stability & efficacy.