Genome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques. In the current study, we developed an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214). As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated. The optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.

中文翻译：

---

使用慢病毒传递系统对鲑鱼细胞系进行高效 CRISPR/Cas9 基因组编辑。


基因组编辑正在改变生物科学研究，但其在非模型生物（例如养殖动物物种）中的应用需要优化。鲑鱼是最重要的水产养殖品种，提高对传染病的遗传抵抗力是一个主要目标。然而，使用基因组编辑来评估细胞系中假定的抗病基因，以及使用全基因组 CRISPR 筛选目前由于缺乏可用的工具和技术而受到限制。在当前的研究中，我们开发了一种使用慢病毒转导的优化方案，将构建体有效整合到奇努克鲑鱼（Oncorhynchus tshwaytcha）细胞系（CHSE-214）的基因组中。作为原理验证，两个目标基因在 EGFP-Cas9 稳定的 CHSE 细胞系中得到了高效编辑；具体来说，外源整合的 EGFP 和内源 RIG-I 基因座。最后，证明了有效使用抗生素选择来丰富已成功编辑的目标人群。本文报道的优化的慢病毒介导的 CRISPR 方法增加了在鲑鱼细胞中进行有效基因组编辑的可能性，特别是对于未来应用全基因组 CRISPR 筛选抗病性。