The continued emergence and spread of highly pathogenic avian influenza viruses (HPAIV), particularly the H5N1, have led to substantial global economic losses and high mortality across a broad range of hosts, including domestic poultry, wild avifauna, mammals, and livestock. The present study evaluates a novel genetic approach using the high fidelity CRISPR/RfxCas13d system – a programmable RNA-targeting genome editing tool – targeting the viral RNA as antiviral strategy against HPAIV in vitro.
Two Tol2 transposon-based plasmids were developed for the stable expression of RfxCas13d and virus-specific guide RNAs (crRNAs) in chicken embryonic fibroblast DF1 cells. The crRNAs were designed to target conserved regions within both the positive- and negative-sense strands of the influenza viral RNA segments. Stable RfxCas13d-expressing DF1 cell lines were subsequently engineered to express either single or multiplexed crRNAs. These modified cell lines were challenged with lab adapted A/WSN/033/H1N1 and various HPAIV strains, including A/Chicken/Lethbridge/9/2020/H7N7, A/Chicken/Vietnam/08/2004/H5N1, and A/Turkey/Indiana/22-003707-003/2022/H5N1 influenza viruses.
Quantitative analysis revealed a statistically significant reduction in viral titres, with crRNAs targeting the positive-sense RNA strand exhibiting greater antiviral activity than those targeting the negative-sense RNA. Mechanistically, we found that the positive sense RNA targeting crRNAs had broader impact in both the positive (copy RNA and mRNA) and negative sense RNA strands (viral genomic RNA) as compared to the negative sense strand targeting crRNA limited only to the negative sense RNA strand (viral genomic RNA). Additionally, the antiviral effect was more pronounced against a slow replicating H7-origin virus relative to a rapidly replicating H5-origin strains. Cell lines expressing multiple crRNAs demonstrated significantly superior viral suppression compared to single-guide counterparts.
Collectively, these findings highlight the potential application of CRISPR/RfxCas13d as the effective antiviral platform for emerging viruses such as the HPAIVs.