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Abbreviations CRDs: cysteine rich domains; ALV: avian leukosis virus; tvb: tumor virus locus B; tva: tumor virus locus A; tvc: tumor virus locus C; W38: tryptophan 38; CRISPR/ Cas9: clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated; DMEM: Dulbecco's minimum essential medium; FBS: fetal bovine serum; ABAM: antibiotic–antimycotic; WL: White Leghorn; CEF: chicken embryonic fbroblast; EDTA: ethylenediaminetetraacetic acid; dpi: days postinfection; SAS: the statistical analysis system; ANOVA: analysis of variance; WT: wild type; GFP: green fuorescent protein; TNFRSF10B: tumor necrosis factor receptor superfamily member 10B; DD: death domain; PAM: protospacer adjacent motif; LTR: long terminal repeat; RCAS: replication-competent ALV LTR with a splice acceptor; HIV: human immunodefciency virus; EIAV: equine infectious anemia virus; FIV: feline immunodefciency virus; RABS: rabies virus; HVEM: herpes virus entry mediator; LDLR: low-density lipoprotein receptor; PGC: primordial germ cell; gRNA: guide RNA. ; Abstract Avian leukosis virus (ALV) is a retrovirus that causes tumors in avian species, and its vertical and horizontal transmission in poultry flocks results in enormous economic losses. Despite the discovery of specific host receptors, there have been few reports on the modulation of viral susceptibility via genetic modification. We therefore engineered acquired resistance to ALV subgroup B using CRISPR/Cas9-mediated genome editing technology in DF-1 chicken fibroblasts. Using this method, we efficiently modified the tumor virus locus B (tvb) gene, encoding the TVB receptor, which is essential for ALV subgroup B entry into host cells. By expanding individual DF-1 clones, we established that artificially generated premature stop codons in the cysteine-rich domain (CRD) of TVB receptor confer resistance to ALV subgroup B. Furthermore, we found that a cysteine residue (C80) of CRD2 plays a crucial role in ALV subgroup B entry. These results suggest that CRISPR/Cas9-mediated genome editing can be used to efficiently modify avian cells and establish novel chicken cell lines with resistance to viral infection. ; This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2015R1A3A2033826). Authors also acknowledge the funding support from the Royal Society International Professorships.