Background: Previously, we showed that targeted disruption of viral receptor genes in avian leukosis virus (ALV) subgroups using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9))-based genome editing confers resistance to ALV subgroups B and J. Here, we used the same strategy to target the receptor expressed by ALV subgroup A (TVA) and generate chicken cells resistant to infection by this virus.
Results: CRISPR/Cas9-based disruption of exon 2 within the tva gene of DF-1 fibroblasts conferred resistance to infection by ALV subgroup A regardless of whether frameshift mutations were introduced during editing. Conversely, overexpression of the wild-type TVA receptor (wtTVA) by tva-modified DF-1 clones restored susceptibility to ALV subgroup A. The results confirm that exon 2, which contains the low-density lipoprotein receptor class A domain of TVA, is critical for virus entry. Furthermore, we sequentially modified DF-1 cells by editing the tva, tvb, and Na+/H+ exchange 1 (chNHE1) genes, which are the specific receptors for ALV subgroups A, B, and J, respectively.
Conclusions: Simultaneous editing of multiple receptors to block infection by different subgroups of ALV confirmed that ALV subgroups A, B, and J do not share host receptors. This strategy could be used to generate cells resistant to multiple viral pathogens that use distinct receptors for cell entry.
Keywords: Avian leukosis virus; CRISPR/Cas9; Genome editing; Host receptor; TVA.