使用 CRISPR/Cas9 技术对种系进行基因编辑使得改变牲畜性状成为可能，包括产生对病毒性疾病的抵抗力。然而，病毒的适应性可能会成为这一努力的主要障碍。最近，通过使用 CRISPR/Cas9 基因组编辑删除 ALV-J 受体 NHE1 内的单个氨基酸 W38，开发出了对禽白血病病毒 J 亚型 (ALV-J) 具有抵抗力的鸡。这种耐药性在体外和体内均得到证实。 W38-/- 鸡胚成纤维细胞对所有测试的 ALV-J 菌株均表现出体外抗性。为了研究 ALV-J 进一步适应的能力，我们使用基于逆转录病毒报告基因的测定来选择适应的 ALV-J 变体。我们假设克服细胞抵抗力的适应性突变将发生在包膜蛋白内。根据这一假设，我们分离并测序了许多适应性病毒变种，并在其包膜基因内发现了八个独立的单核苷酸取代。为了确认这些替换的适应能力，我们将它们引入到原始的逆转录病毒报告基因中。所有八种变体在体外都能在 W38-/- 鸡胚胎成纤维细胞中有效复制，而在体内，W38-/- 鸡对其中两种变体诱导的肿瘤敏感。重要的是，经过更广泛修饰的受体等位基因仍然对病毒具有抵抗力。这些结果证明了牲畜基因组工程中抗病毒抗性的重要策略，并说明由较小的受体修饰引起的细胞抗性可以通过适应的病毒变体来克服。我们的结论是，需要更复杂的编辑才能获得强大的抵抗力。版权所有：© 2024 Matoušková 等人。这是一篇根据知识共享署名许可条款分发的开放获取文章，允许在任何媒体上不受限制地使用、分发和复制，前提是注明原始作者和来源。

Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.Copyright: © 2024 Matoušková et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.