This review systematically summarize recent progress in CRISPR/Cas9-based screening studies of major livestock and poultry viruses, including foot-and-mouth disease virus (FMDV), swine enteric coronaviruses, African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), avian leukosis virus (ALV), and other zoonotic pathogens.
Abstract
Viral infectious diseases in livestock and poultry cause substantial economic losses worldwide and pose persistent zoonotic threats to public health. Elucidating virus-host interactions is essential for understanding viral pathogenesis and developing effective control strategies. In recent years, genome-wide CRISPR/Cas9 functional screening has emerged as a powerful and unbiased high-throughput approach for identifying host dependency and restriction factors. Enabled by the development of species-specific sgRNA libraries, this technology has significantly advanced research in veterinary virology. In this review, we systematically summarize recent progress in CRISPR/Cas9-based screening studies of major livestock and poultry viruses, including foot-and-mouth disease virus (FMDV), swine enteric coronaviruses, African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), avian leukosis virus (ALV), and other zoonotic pathogens. We highlight key host factors involved in viral entry, replication, and egress, and integrate these findings to delineate conserved cross-viral dependency networks, such as sialic acid biosynthesis, endosomal–lysosomal trafficking, double-membrane vesicle (DMV) formation, and interferon signaling pathways. Furthermore, we discuss the translational potential of these genomic discoveries for practical agricultural applications, particularly in the development of host-targeted broad-spectrum antivirals and the generation of disease-resistant livestock (e.g., receptor-edited pigs and chickens) through precise genome editing. Finally, we outline future perspectives, including the integration of single-cell transcriptomics and in vivo validation, thereby providing a comprehensive framework for advancing disease control and sustainable breeding in animal agriculture.
Background Foot-and-mouth disease virus (FMDV) causes substantial economic losses in global livestock production; however, the key host factors supporting its early infection process remain poorly characterized. Methods In this study, we performed an unbiased genome-wide CRISPR/Cas9 knockout screening using porcine cells to screen and identify host factors involved in FMDV infection. Results We identified that the E3 ubiquitin ligase RNF24 supports efficient FMDV entry. RNF24 depletion inhibits viral entry and replication, whereas its overexpression enhances viral infectivity. Mechanistically, RNF24 preferentially promotes K27-linked non-degradative polyubiquitination of leupaxin (LPXN) at lysine 162, driving LPXN’s trafficking to the plasma membrane. At the membrane, LPXN assembles a ternary integrin-LPXN-VP1 complex that strengthens virus-receptor interactions and promotes viral adsorption and entry. Disruption of this ubiquitination event via the LPXN K162R mutation impairs complex formation and compromises viral entry. Conclusion Together, our study reveals a ubiquitin-dependent RNF24-LPXN regulatory axis that supports FMDV entry, highlights the role of non-degradative ubiquitination in viral pathogenesis, and proposes this interface as a potential target for antiviral intervention.
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This review provides an overview of recent advances in the development of genetically engineered pigs with enhanced resistance to major viral diseases, focusing on targeted gene modifications using knockout, knock-in, and overexpression approaches.
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By systematically removing host restriction factors, this platform provides a versatile and powerful strategy for accelerating viral propagation, offering a strong foundation for more efficient development and large-scale production of cell culture-based anti-viral vaccines.
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BACKGROUND
Bovine viral diarrhea virus (BVDV) is a viral pathogen that may cause reproductive disorders, health, and performance. BVDV serotypes have undergone dynamic changes due to sustained genetic diversity, particularly in BVDV type 1 (BVDV1), which has compromised the specificity and sensitivity of diagnostic assays. Furthermore, BVDV1 is prevalent worldwide, causing significant losses to the livestock industry. Therefore, in order to further enhance the control and elimination of BVDV, it is necessary to detect and prevent the spread of BVDV. Therefore, establishing a faster and more accurate on-site detection method is particularly important.
METHODS
Traditional PCR and ELISA methods are limited by cost, sensitivity, and equipment dependence. In this study, we designed primers based on the conserved 5'Untranslated Region (5'UTR) of BVDV1 and developed a method that combines one-step reverse transcription recombinase assisted amplification (RT-RAA) and CRISPR/Cas12a based fluorescent substrates in a mixture to quickly screen and accurately distinguish BVDV1 for the diagnosis and differentiation of common bovine viruses such as Bovine Coronavirus (BCoV) and Bovine Herpesvirus type 1 (BoHV) et al. The established RT-RAA-CRISPR/Cas12a scheme can achieve the entire inspection process in a portable metal bath at 40℃. We further optimized the reaction conditions of RT-RAA-CRISPR/Cas12a to detect its sensitivity, specificity, and repeatability. The established RT-RAA-CRISPR/Cas12a and conventional PCR were used to detect 300 clinical samples of cattle, verifying the actual detection ability.
RESULTS
This method did not show any cross-reactivity with the other nine common bovine viruses, demonstrating excellent specificity. The minimum template concentration required to trigger significant trans cleavage activity without RT-RAA amplification is 10 copies/µL of BVDV template. However, combined with RT‑RAA amplification, the detection limit was significantly decreased to one copy of the BVDV template per reaction. In simulated sample testing, this method achieved 100% accuracy, and the entire detection process can be completed within 30 min. In clinical sample testing, RT-RAA-CRISPR/Cas12a showed a higher sensitivity rate for BVDV1, as the positive detection rate was 1.85 times higher than that of conventional PCR.
CONCLUSION
In summary, the BVDV-1 RT-RAA-CRISPR/Cas12a detection method established in this study offers several advantages, including fast operation, high sensitivity, strong targeting, and good repeatability. Moreover, the same test tube reaction system makes it widely applicable in resource-limited environments, making it of great value in large-scale screening during BVDV outbreaks. This method not only expands the diagnostic toolbox of BVDV but also provides an up-and-coming solution for controlling the spread of BVDV in cattle herds.
