CRISPR/Cas12a-Based Rapid Diagnostic Test for High-Risk HPV: Development, Evaluation, and Genotyping in Clinical Specimens

High-risk human papillomavirus (HR-HPV) infections pose a significant global health threat, being the primary cause of cervical cancer. Approximately 604,126 new cases of cervical cancer were reported worldwide in 2020, with the majority of deaths occurring in low-and middle-income countries. Early and accurate detection of HR-HPV is crucial for preventing the progression to cervical cancer. Traditional detection methods, such as real-time PCR, have limitations in terms of complexity, cost, and accessibility, especially in resource-constrained settings. The emergence of CRISPR-Cas12a technology offers a revolutionary approach to address these challenges.

Fig.1 Illustration of the CRISPR/Cas12a-based fluorescent assay for HPV detection in clinical samples. (Yin L., et al., 2025)

The Menace of HR-HPV

HPV Types and Cancer Link

There are over 200 known types of HPV, with about 40 associated with genital tract infections. Among them, 14 types are classified as high-risk (HR-HPV), including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Persistent infection with HR-HPV is closely linked to the development of cervical cancer. For instance, HPV 16 and 18 alone are responsible for nearly 70% of cervical cancer cases, while HPV 45 and 31 contribute to about 5% and 10% respectively. In addition to cervical cancer, HR-HPV is also associated with 90% of anal cancers, 65% of vaginal cancers, 50% of vulvar cancers, 35% of penile cancers, and 60% of oropharyngeal cancers.

Inadequacies of Current Detection Methods

Current gold-standard detection methods like real-time PCR require sophisticated equipment, trained personnel, and are relatively time-consuming. For example, the Cobas HPV test, which is widely used, involves a complex workflow and high costs. This makes it difficult to implement in areas with limited healthcare resources, where the need for HPV screening is most urgent. There is an unmet need for a simple, rapid, sensitive, and affordable alternative for HR-HPV detection.

Understanding CRISPR-Cas12a Technology

The CRISPR-Cas System Basics

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated proteins) systems are part of the adaptive immune defenses in bacteria and archaea. The system consists of CRISPR arrays (repeats and spacers) and Cas proteins. When a bacterium is invaded by a virus or plasmid, the CRISPR system captures a piece of the invader's DNA (spacer) and incorporates it into its own CRISPR array. This is the adaptation phase. In the expression phase, the CRISPR array is transcribed and processed into crRNAs (CRISPR RNAs). In the interference phase, the crRNA guides the Cas protein to recognize and cleave the complementary DNA of the invading pathogen.

CRISPR-Cas12a in Particular

CRISPR-Cas12a is a type V CRISPR-Cas system. It uses a single crRNA for target recognition. Once Cas12a binds to the target DNA sequence (guided by the crRNA) that has a specific protospacer adjacent motif (PAM, 5'-TTN for FnCas12a), it not only cleaves the target DNA but also exhibits trans-cleavage activity. This means it can non-specifically cleave single-stranded DNA (ssDNA) reporters, which is exploited in diagnostic assays.

The CRISPR-Cas12a-Based Diagnostic Platform for HR-HPV

Combining with MIRA for Amplification

In a recent study, researchers combined CRISPR-Cas12a with Multienzyme Isothermal Rapid Amplification (MIRA). MIRA is an isothermal amplification technique that can amplify DNA at a constant temperature (39-42°C). This combination creates a two-step process for HR-HPV detection. First, MIRA rapidly amplifies the target HPV DNA in a single tube, even when the initial DNA concentration is very low. Then, the amplified DNA is recognized by the CRISPR-Cas12a system. The Cas12a, guided by type-specific crRNAs, binds to the amplified HPV DNA. Once bound, the trans-cleavage activity of Cas12a is activated, cleaving fluorescently-labeled ssDNA reporters.

Key Features of the Platform

Exceptional Sensitivity: The assay can detect as few as 2 copies of HPV DNA per microliter. In clinical trials, it outperformed traditional qPCR in detecting low-concentration samples. For example, in a test with 258 cervical swab samples, it correctly identified all positive cases, including those with very low levels of HPV DNA.
High Specificity: The platform shows 100% specificity. Through a "14×14 matrix test", it was confirmed that each crRNA specifically targets its corresponding HPV type, with no cross-reactivity. This ensures that false positives are minimized.
Multiplexed Detection: It enables the detection of all 14 HR-HPV types in a single-tube assay. This is a significant improvement over previous CRISPR-based methods that could typically detect only 2-13 types. The use of a crRNA pool allows for the simultaneous targeting of multiple HPV types.
Rapid Results: The entire process takes only 30 minutes, in contrast to qPCR, which can take hours. This speed is invaluable for point-of-care testing, allowing for immediate screening and triage.
Visual Readout: The fluorescence signals can be measured using a microplate reader or even visualized by the naked eye under UV or blue light. This eliminates the need for expensive and complex equipment, making the test suitable for use in resource-poor settings, such as rural clinics or mobile screening units.

Clinical Validation and Impact

Comparison with Existing Tests

In a comparison with the Cobas HPV test (qPCR), the CRISPR-Cas12a-based assay demonstrated superior sensitivity. While the Cobas test showed 97.9% sensitivity for HPV-16 and 95.6% for HPV-18, the CRISPR-based method achieved 100% sensitivity for all 14 HR-HPV types. This improved sensitivity is crucial as it can detect low-level infections that may be missed by qPCR, potentially preventing undiagnosed cases from progressing to cancer.

Real-World Applicability

The simplicity of the CRISPR-Cas12a platform reduces the need for highly trained personnel. Healthcare workers with minimal training can perform the test, which expands the screening capacity, especially in regions with a shortage of skilled healthcare providers. Additionally, the lower cost of the assay, due to reduced equipment and reagent requirements, makes it more affordable for large-scale screening programs.

Future Prospects and Challenges

Potential Applications

Global Cervical Cancer Screening: The platform has the potential to be a game-changer in global cervical cancer screening, especially in low-resource countries where 85% of cervical cancer deaths occur. It can be easily integrated into existing healthcare programs, providing an accessible and accurate screening tool.
Point-of-Care Testing: For routine check-ups or in emergency situations, the rapid and easy-to-use nature of the test makes it ideal for point-of-care testing. This can lead to earlier detection and intervention.
Cost-Effective Screening: The lower cost of the CRISPR-Cas12a-based assay compared to traditional methods can make HPV screening more affordable, potentially increasing the coverage of screening programs.

Challenges Ahead

Sample Optimization: Although the assay has shown high performance with cervical swab samples, further optimization is needed for other types of samples, such as self-collected samples. This would increase the convenience and acceptance of the screening process.
Workflow Integration: Integrating the new assay into existing healthcare workflows may pose challenges. There is a need to ensure seamless communication between different healthcare providers and departments to ensure the effective implementation of the screening process.
Larger-Scale Validation: While the initial clinical trials have shown promising results, larger-scale validation in diverse populations is required to fully establish the reliability and effectiveness of the CRISPR-Cas12a platform.

Conclusion

CRISPR-Cas12a technology represents a significant leap forward in the detection and genotyping of high-risk HPV. By addressing the limitations of traditional detection methods, it offers a rapid, sensitive, specific, and accessible solution for HR-HPV screening. As research continues to optimize and validate this technology, it has the potential to play a pivotal role in global efforts to reduce the burden of cervical cancer and other HPV-related diseases. With further development and implementation, CRISPR-Cas12a could be the key to achieving the World Health Organization's goal of reducing cervical cancer incidence to below 4 cases per 100,000 women by 2030.

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Reference

Yin, Lijuan, et al. "Development and evaluation of a CRISPR/Cas12a-based diagnostic test for rapid detection and genotyping of HR-HPV in clinical specimens." Microbiology Spectrum 13.1 (2025): e02253-24.

This article is for research use only. Do not use in any diagnostic or therapeutic application.