Application of CRISPR-Cas System in Human Papillomavirus Detection Using Biosensor Devices and Point-of-Care Technologies

Abstract

Human papillomavirus (HPV) is the most common virus for genital tract infections. Cervical cancer ranks as the fourth most prevalent cancer globally, with over 99% of cases in women attributed to HPV infection. This infection continues to pose an ongoing threat to public health. Therefore, the development of rapid, high-throughput, and sensitive HPV detection platforms is important, especially in regions with limited access to advanced medical resources. CRISPR-based biosensors, a promising new method for nucleic acid detection, are now rapidly and widely used in basic and applied research and have received much attention in recent years for HPV diagnosis and treatment. In this review, we discuss the mechanisms and functions of the CRISPR-Cas system, focusing on its applications in HPV diagnostics. The review covers CRISPR technologies such as CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas13, along with nucleic acid amplification methods, CRISPR-based signal output systems, and point-of-care testing (POCT) strategies. This comprehensive overview highlights the versatility and potential of CRISPR technologies in HPV detection. We also discuss the numerous CRISPR biosensors developed since the introduction of CRISPR to detect HPV. Finally, we discuss some of the challenges faced in HPV detection by the CRISPR-Cas system.

Fig. 1.

CRISPR-based HPV detection systems utilize Cas9 for specific HPV DNA binding, Cas12 for collateral cleavage of DNA, and Cas13 for targeting and cleaving HPV RNA, enabling robust and versatile diagnostics.

Fig. 2.

(A) Schematic illustration of CRISPR-Cas12a-based ECL biodetection toward HPV16 [74]. Copyright 2022, American Chemical Society. (B) Schematic illustration of DNA tetrahedron nanostructure and the trans-cleavage activity of CRISPR/Cas12a-mediated ECL biosensor [70]. Copyright 2023, American Chemical Society. Schematic diagram illustrating the strategy to detect viral DNA using SERS-active graphene oxide (GO)/triangle Au nanoflower (GO-TANF). (C) Enhanced SERS signaling. (D) Reverse cleavage effect of CRISPR-Cas12a. (E) ssDNA breakage [85]. Copyright 2021, American Chemical Society.

Fig. 3.

Working principle of the detection platform. (A) Overview of the steps involved in the detection process in M-D3. (B) On-chip assay [90]. Copyright 2023, American Chemical Society. Brief overview of the steps involved in the subtyping process. (C) Sample nucleic acid extraction and amplification. (D) On-chip testing principles [99]. Copyright 2022, Springer Nature.

Fig. 4.

LFD-based detection of HPV16 and HPV18 plasmids and in patient samples. (A) Scheme showing the mechanism of the LFD-based readout. (B) Testing of the amplified HPV16/18 plasmids with LFDs. (C) On-chip testing of HPV16 and HPV18 in clinical samples based on the LFD readout [94]. Copyright 2023, American Chemical Society.

Fig. 5.

(A and B) Schematic diagram of CRISPR-Cas12a colorimetric detection of HPV16 [104]. Copyright 2024, Elsevier. (C) Schematic of a CRISPR-based DNA biosensor for high-sensitivity colorimetric viral DNA detection using magnetic enzyme complexes [109]. Copyright 2024, MDPI.

Fig. 6.

(A) Detection schematic diagram of the method based on CRISPR/dCas9-assisted HRP-catalyzed signal amplification [110]. Copyright 2023, American Chemical Society. (B) Schematic diagram of the CRISPR/Cas9 workflow and detection of genes for HPV16 and HPV18 as proof of principle [113]. Copyright 2018, Springer Nature.

Fig. 7.

(A) Orthogonal Cas12a/Cas13a detection enables dual-color fluorescence visualization of HPV16/18 through target-specific cleavage of DNA and RNA reporters [95]. Copyright 2022, RSC Pub. (B) Multiplexed one-pot CRISPR detection integrates amplification, thermostable CRISPR enzymes, and signal interpretation for HPV diagnostics [116]. Copyright 2024, American Chemical Society.