Dhakal, S.; Smith, A. J.; Weiss, E.; Islam, Z. M.; Nazareth, L.; Lee, T.; Gough, T.; Nair, K. K.; Wilson, L.; Wynne, J. W.; Jenkins, K.; Challagulla, A.

Highly pathogenic avian influenza viruses (HPAIVs) continue to cause substantial disease in birds and mammals, with repeated H5N1 spillovers highlighting the need for broadly protective antiviral strategies. Here we develop a programmable RNA-targeting antiviral platform based on RfxCas13d and evaluate its activity in avian cells. Screening of five Cas13 orthologs in chicken DF1 fibroblasts revealed RfxCas13d as the most potent and well tolerated effector. Virus-specific CRISPR RNAs (crRNAs) targeting conserved regions of positive- and negative-sense influenza RNA were tested against A/WSN/033[H1N1] and multiple HPAIV isolates, including a member of clade 2.3.4.4b H5N1. Targeting positive-sense RNA conferred superior influenza inhibitory activity and further enhanced by multiplexed crRNA expression. These findings establish RfxCas13d as a versatile RNA-guided antiviral platform and provide a route for broad-spectrum influenza control through conserved RNA targeting.