CRISPR-Cas12a Based Biosensor for Rapid Detection of African Swine Fever Virus in Porcine Blood

Introduction

African swine fever virus (ASFV) is a large, enveloped, double-stranded DNA virus belonging to the family Asfarviridae, genus Asfivirus [1]. ASFV causes a highly contagious and often fatal hemorrhagic disease in domestic swine and wild boar, with mortality rates approaching 100% in acute infections [1, 2]. The virus is endemic in sub-Saharan Africa, Sardinia, and parts of Eastern Europe and Asia, and its continued spread poses a severe threat to global swine production and food security [2]. Rapid and accurate detection of ASFV is critical for implementing control measures such as quarantine, culling, and movement restrictions [3]. Conventional diagnostic methods include virus isolation, antigen detection by enzyme-linked immunosorbent assay (ELISA), and nucleic acid amplification by quantitative polymerase chain reaction (qPCR) [3, 4]. While qPCR remains the gold standard for its high sensitivity and specificity, it requires expensive thermal cycling equipment, trained personnel, and a well-equipped laboratory, limiting its utility in field settings and resource-limited regions [4, 5].

The emergence of CRISPR-based diagnostic platforms has opened new avenues for rapid, sensitive, and portable nucleic acid detection [5]. CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease that, upon specific recognition of a target DNA sequence, activates non-specific single-stranded DNase (ssDNase) activity, a phenomenon termed collateral cleavage [6]. This collateral activity can be harnessed to cleave reporter molecules, generating a detectable signal [6]. When combined with isothermal amplification methods such as recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP), CRISPR-Cas12a biosensors can achieve attomolar sensitivity without the need for thermal cycling [7]. This article describes the development and validation of a CRISPR-Cas12a-based biosensor targeting the ASFV p72 gene for rapid detection of the virus in porcine blood, highlighting its potential for point-of-care (POC) deployment.

Mechanism of CRISPR-Cas12a Collateral Cleavage

CRISPR-Cas12a is a class 2, type V CRISPR effector protein that recognizes a short protospacer adjacent motif (PAM) sequence (typically 5'-TTTV-3' for AsCas12a) and uses a CRISPR RNA (crRNA) to guide sequence-specific binding to complementary double-stranded DNA (dsDNA) targets [6, 8]. Upon target binding, Cas12a undergoes a conformational change that activates its DNase active site, leading to site-specific cleavage of the target DNA [8]. Importantly, this activation also triggers robust non-specific single-stranded DNase activity, which degrades any single-stranded DNA molecules in the vicinity [6]. This collateral cleavage can be monitored using fluorophore-quencher (FQ) reporter probes: intact probes are quenched, but cleavage by activated Cas12a separates the fluorophore from the quencher, producing a fluorescence signal [6, 7]. The signal intensity is proportional to the amount of target DNA present, enabling quantitative or semi-quantitative detection [7].

The collateral cleavage mechanism is distinct from that of Cas13a, which targets RNA and exhibits RNase collateral activity [5]. Cas12a's preference for DNA targets makes it particularly suitable for detecting DNA viruses such as ASFV [5, 6]. The reaction can be performed at a constant temperature (typically 37-42 degrees Celsius), eliminating the need for thermal cyclers [7].

Assay Design: Targeting the ASFV p72 Gene

The p72 gene (also designated B646L) encodes the major capsid protein of ASFV and is highly conserved across all known ASFV genotypes [1, 9]. This genetic stability makes p72 an ideal target for molecular diagnostics, as it minimizes the risk of false negatives due to sequence variation [9]. The CRISPR-Cas12a biosensor described here employs a crRNA designed to recognize a conserved region within the p72 gene. The crRNA includes a 20-24 nucleotide spacer sequence complementary to the target, preceded by a PAM sequence (5'-TTTV-3') in the target DNA [6, 8]. Multiple crRNA candidates are typically screened in silico and validated experimentally to select the one with the highest specificity and sensitivity [10].

The assay workflow consists of three main steps: (1) nucleic acid extraction from porcine blood samples, (2) isothermal amplification of the p72 target region, and (3) CRISPR-Cas12a collateral cleavage detection. A schematic representation is provided in Figure 1.

flowchart TD
    A[Porcine blood sample], > B[DNA extraction]
    B, > C[Isothermal amplification (RPA or LAMP)]
    C, > D[CRISPR-Cas12a detection reaction]
    D, > E[Add FQ reporter probe]
    E, > F[Incubate at 37-42°C]
    F, > G[Measure fluorescence or lateral flow signal]
    G, > H{Signal above threshold?}
    H, >|Yes| I[ASFV positive]
    H, >|No| J[ASFV negative]

Figure 1. Workflow of the CRISPR-Cas12a biosensor for ASFV detection in porcine blood.

Isothermal Amplification: RPA and LAMP

Isothermal amplification is essential to achieve the high sensitivity required for direct detection of viral DNA from clinical samples, as the collateral cleavage signal from unamplified target is often insufficient [7]. Two commonly used isothermal methods are recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) [7, 11].

RPA employs recombinase enzymes to pair primers with homologous sequences in the template DNA, followed by strand displacement synthesis by a polymerase, all at a constant temperature of 37-42 degrees Celsius [11]. RPA is rapid, typically producing detectable amplicons within 10-20 minutes, and is tolerant to inhibitors present in crude lysates [11]. For ASFV detection, RPA primers targeting the p72 gene are designed to generate an amplicon of 100-200 base pairs, which is optimal for Cas12a recognition [7, 12].

LAMP uses four to six primers recognizing six to eight distinct regions on the target DNA, and a strand-displacing polymerase (e.g., Bst polymerase) to produce stem-loop structures with multiple amplification products [13]. LAMP operates at 60-65 degrees Celsius and can generate up to 10^9 copies within 30-60 minutes [13]. LAMP is highly specific due to the multiple primer binding requirements, but primer design is more complex [13]. Both RPA and LAMP can be performed with simple heating blocks or water baths, making them suitable for field use [7, 13].

Collateral Cleavage Detection and Signal Readout

After isothermal amplification, the amplicons are mixed with the Cas12a-crRNA complex and an FQ reporter probe (e.g., a 5-nucleotide ssDNA with a fluorophore and quencher at opposite ends) [6, 7]. The reaction is incubated at 37-42 degrees Celsius for 5-30 minutes. Activated Cas12a cleaves the reporter probe, releasing fluorescence that can be measured using a portable fluorometer or a smartphone-based reader [7, 14]. Alternatively, the collateral cleavage can be coupled to a lateral flow strip by using a biotinylated reporter and anti-fluorescein antibodies, enabling visual readout without instrumentation [14]. This dual readout capability enhances the versatility of the biosensor for POC applications [14].

Analytical Sensitivity and Specificity

The analytical sensitivity of the CRISPR-Cas12a biosensor for ASFV is typically determined using serial dilutions of synthetic p72 gene fragments or quantified viral genomic DNA [7, 12]. Studies have reported limits of detection (LOD) as low as 1-10 copies per reaction when combined with RPA or LAMP, which is comparable to or better than conventional qPCR [7, 12]. For example, an RPA-Cas12a assay targeting the p72 gene achieved an LOD of 2 copies per microliter in spiked porcine blood samples [12]. The dynamic range spans at least 5-6 orders of magnitude, allowing both qualitative and semi-quantitative detection [7].

Specificity is assessed by testing the assay against other swine pathogens, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and swine influenza A virus (SIV) [7, 12]. The crRNA is designed to have no significant homology to these non-target genomes, and experimental results confirm no cross-reactivity [7, 12]. Additionally, the assay can differentiate ASFV from other hemorrhagic fever viruses of swine, such as CSFV, which is caused by a different virus family (Flaviviridae) [2, 3].

Comparison with Quantitative PCR

Quantitative PCR (qPCR) remains the reference standard for ASFV detection due to its high sensitivity and established protocols [4]. However, qPCR requires thermal cycling equipment, which is expensive and not readily available in many field settings [4]. The CRISPR-Cas12a biosensor offers several advantages:

• Speed: Total assay time from sample to result is typically 30-60 minutes, compared to 2-3 hours for qPCR [7, 12].
• Temperature: Isothermal amplification and Cas12a detection occur at a single constant temperature, eliminating the need for thermal cyclers [7].
• Portability: The reaction can be performed with minimal equipment (e.g., a heat block and a fluorometer or lateral flow strip) [14].
• Cost: Reagent costs are lower than those for qPCR, especially when using in-house produced Cas12a protein [7].

Disadvantages include the potential for false positives due to amplicon contamination (common to all amplification-based methods) and the need for careful primer and crRNA design to avoid off-target activation [7]. Additionally, qPCR provides absolute quantification through standard curves, whereas CRISPR-based methods are typically semi-quantitative [4, 7].

Point-of-Care Deployment and Field Validation

The CRISPR-Cas12a biosensor has been validated using spiked and naturally infected porcine blood samples [7, 12]. Sample preparation involves simple DNA extraction using commercial kits or rapid boiling methods [12]. The entire assay can be performed in a suitcase-sized portable laboratory, making it suitable for on-farm testing, border inspection, and outbreak surveillance [14]. Field studies have demonstrated concordance rates of >95% with qPCR results, with no significant difference in sensitivity [12].

Integration with lateral flow readout further simplifies the workflow, as results can be interpreted visually without instrumentation [14]. This is particularly valuable in low-resource settings where electricity or trained personnel may be limited [14]. The biosensor can also be adapted for detection of ASFV in oral fluids, which are easier to collect than blood, although blood samples typically yield higher viral loads [15].

Conclusion

The CRISPR-Cas12a-based biosensor targeting the ASFV p72 gene represents a significant advancement in rapid veterinary diagnostics. By combining isothermal amplification with Cas12a collateral cleavage, the assay achieves sensitivity and specificity comparable to qPCR while offering faster turnaround times, lower equipment requirements, and compatibility with POC formats. This technology has the potential to enhance surveillance and control efforts for African swine fever, particularly in regions where laboratory infrastructure is limited. Future developments may include multiplexed detection of multiple swine pathogens, integration with microfluidic devices, and further optimization for direct detection from crude samples without extraction. The biosensor aligns with broader trends in veterinary molecular diagnostics toward decentralized, rapid, and user-friendly testing platforms.

References

[1] Dixon LK, Chapman DAG, Netherton CL, Upton C. African swine fever virus. In: Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW, Zhang J, editors. Diseases of Swine. 11th ed. Wiley-Blackwell; 2019. p. 443-452.

[2] Sánchez-Vizcaíno JM, Mur L, Gómez-Villamandos JC, Carrasco L. African swine fever: an epidemiological update. Transboundary and Emerging Diseases. 2012;59(Suppl 1):27-35.

[3] Oura CAL, Edwards L, Batten CA. Virological diagnosis of African swine fever: comparative study of available tests. Virus Research. 2013;173(1):150-158.

[4] King DP, Reid SM, Hutchings GH, Grierson SS, Wilkinson PJ, Dixon LK, et al. Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus. Journal of Virological Methods. 2003;107(1):53-61.

[5] Chertow DS. Next-generation diagnostics with CRISPR. Science. 2018;360(6387):381-382.

[6] Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018;360(6387):436-439.

[7] Broughton JP, Deng X, Yu G, Fasching CL, Servellita V, Singh J, et al. CRISPR-Cas12a-based detection of SARS-CoV-2. Nature Biotechnology. 2020;38(7):870-874.

[8] Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163(3):759-771.

[9] Bastos ADS, Penrith ML, Crucière C, Edrich JL, Hutchings G, Roger F, et al. Genotyping field strains of African swine fever virus by partial p72 gene characterisation. Archives of Virology. 2003;148(4):693-706.

[10] Labun K, Montague TG, Gagnon JA, Thyme SB, Valen E. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research. 2016;44(W1):W272-W276.

[11] Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA detection using recombination proteins. PLoS Biology. 2006;4(7):e204.

[12] Wang X, Ji P, Fan H, Dang L, Wan W, Liu S, et al. CRISPR/Cas12a technology combined with recombinase polymerase amplification for rapid and sensitive detection of African swine fever virus. Transboundary and Emerging Diseases. 2021;68(4):2064-2073.

[13] Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research. 2000;28(12):e63.

[14] Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018;360(6387):439-444.

[15] Mur L, Gallardo C, Soler A, Zimmermann J, Pelayo V, Nieto R, et al. Potential use of oral fluid samples for serological diagnosis of African swine fever. Veterinary Microbiology. 2013;165(1-2):135-139. *** Disclaimer: This article is for educational and informational purposes only. It is not intended to substitute for professional veterinary advice, diagnosis, treatment, or regulatory guidance. Always consult a licensed veterinarian or qualified specialist regarding animal health, disease diagnosis, and therapeutic decisions.