CRISPR diagnostics transform monkeypox detection for global marketsCRISPR diagnostics transform monkeypox detection for global markets

The re-emergence and global dissemination of the monkeypox virus (MPXV), a double-stranded DNA virus belonging to the Orthopoxvirus genus in the Poxviridae family, has underscored the need for rapid, accurate, and accessible diagnostic tools. The 2022 multi-country outbreaks prompted the World Health Organization (WHO) to declare monkeypox a Public Health Emergency of International Concern (PHEIC) on July 23, 2022 (Rabaan et al., 2023). Historically endemic to Central and West Africa, MPXV has now been identified in numerous non-endemic regions, revealing critical gaps in global diagnostic capabilities (Alakunle et al., 2020).

Clinically, monkeypox presents with signs and symptoms that closely mimic other infectious diseases, such as those caused by herpes simplex virus, molluscum contagiosum virus, measles virus, enteroviruses, varicella-zoster virus, and several bacterial skin infections (da Silva et al., 2023). These similarities complicate clinical diagnosis, necessitating laboratory confirmation for accurate differential diagnosis and timely public health response.

Conventional diagnostic approaches for MPXV include viral culture, electron microscopy, serological assays, loop-mediated isothermal amplification (LAMP), whole genome sequencing, and real-time polymerase chain reaction (PCR) (Fan et al., 2024; da Silva et al., 2023). Among these, real-time PCR is considered the gold standard due to its high sensitivity and specificity (WHO, 2022). However, PCR platforms require sophisticated infrastructure and trained personnel, limiting their utility in low-resource settings where MPXV is often endemic (Yu et al., 2024).

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The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated Cas proteins has revolutionised molecular biology, with significant implications for diagnostics. Originally identified as a component of bacterial adaptive immunity (Kostyusheva et al., 2022), the CRISPR-Cas system has been extensively repurposed for genome editing and, more recently, for molecular diagnostics. CRISPR-based diagnostics utilise programmable Cas enzymes (e.g., Cas12, Cas13) guided by RNA sequences to recognise and cleave specific nucleic acid targets. Upon target recognition, collateral cleavage of reporter molecules generates a detectable signal, often read via fluorescence or lateral flow assays (Kaminski et al., 2021).

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Pioneering platforms such as SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) (Cas13a-based) and DETECTR (DNA endonuclease-targeted CRISPR trans reporter) (Cas12a-based) have demonstrated rapid and accurate detection of several viral pathogens, including SARS-CoV-2, Ebola, Zika, and Dengue viruses (Gootenberg et al., 2018; Broughton et al., 2020). These systems combine isothermal amplification techniques, such as recombinase polymerase amplification (RPA), with CRISPR-based detection to achieve high sensitivity and specificity without the need for thermal cyclers, thus enhancing field-deployability.

Recent studies have extended CRISPR-based diagnostics to MPXV detection. Sui et al. (2022) reported a Cas12a-based assay capable of detecting MPXV DNA within two to 10 minutes using a fluorescent readout, demonstrating its potential for point-of-care (POC) application. Singh et al. (2022) designed a similar CRISPR-Cas12a system that exhibited high specificity in detecting synthetic MPXV DNA. Yu et al. (2024) further developed a single-step RPA-CRISPR/Cas12a assay that detected MPXV within 40 minutes with a limit of detection (LOD) of 1 copy/μL, showing 100 per cent concordance with standard PCR.

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Similarly, Low et al. (2023) validated an isothermal amplification CRISPR-Cas12a assay with comparable sensitivity and specificity to PCR, achieving an LOD of 1 copy/μL in 45 minutes. Wang et al. (2024) introduced the SCOPE (Streamlined CRISPR On Pod Evaluation) platform, which targets the A27L gene of MPXV and detects viral DNA at concentrations as low as 0.5 copies/μL in under 15 minutes, further demonstrating the platform’s applicability in resource-limited environments.

CRISPR-based diagnostics offer several advantages over conventional methods. These include rapid turnaround times, minimal equipment requirements, high analytical performance, potential for multiplexing, and adaptability for field use. Additionally, the low cost and ease of use position CRISPR diagnostics as a transformative technology for public health surveillance and outbreak response, particularly in underserved regions. Importantly, these platforms have the potential to bridge the existing diagnostic gap in endemic areas by facilitating early detection, prompt treatment, and timely containment of outbreaks.

In conclusion, the integration of CRISPR-based diagnostics into MPXV detection strategies holds significant promise for enhancing global health preparedness. With performance metrics comparable to or exceeding those of PCR, yet with reduced infrastructural demands, CRISPR diagnostics represent a vital innovation for point-of-care detection of emerging infectious diseases such as monkeypox.

As research continues to refine and validate these tools, their widespread deployment could significantly improve diagnostic capacity, particularly in low-resource settings where timely and accurate testing is most needed.

*Credits:

Timothy Nathaniel Yakubu, DauLab BioGenomics & Biomedical Services, Kaduna, Nigeria, and Global Health and Infectious Disease Control Institute, Nasarawa State University Keffi, Nigeria.

Adamu Ishaku Akyla, Global Health and Infectious Disease Control Institute, Nasarawa State University Keffi, Nigeria.

References available on request.