Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes.
Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes.
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DOI：
10.1007/s10126-020-09995-y
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发表时间：
2020-10
期刊：
影响因子：
--
通讯作者：
Houston RD
中科院分区：
文献类型：
--
作者：
Gratacap RL;Jin YH;Mantsopoulou M;Houston RD
传染病和寄生虫病对鲑鱼物种的水产养殖具有重大的负面经济和动物福利影响。提高对这些疾病的宿主反应和遗传抗性的功能基础的了解是制定预防和治疗方案的关键。细胞系为研究鲑鱼传染病提供了有价值的模型，使用 CRISPR/Cas 系统进行基因组编辑为评估这些系统中特定基因的功能提供了令人兴奋的途径。虽然 CRISPR/Cas 编辑已在奇努克鲑鱼细胞系 (CHSE-214) 中成功进行，但迄今为止还没有编辑来自最具商业相关性的鲑鱼物种大西洋鲑鱼和虹鳟鱼的细胞系的报道，这些细胞难以转导，因此使用慢病毒介导的方法进行编辑。在当前的研究中，使用核糖核蛋白（RNP）复合物对鲑科鱼细胞系进行基因组编辑的方法进行了优化，并在最常用的鲑鱼细胞系中进行了测试：大西洋鲑鱼（SHK-1和ASK细胞系）、虹鳟鱼（RTG-2）和奇努克鲑鱼（CHSE-214）。基于 Cas9 或 Cas12a 的 RNP 电穿孔可有效地对所有测试细胞系（通常超过 90% 的细胞进行编辑）进行靶向编辑，并且酶的选择扩大了这些物种基因组内用于编辑的潜在靶位点的数量。这些优化的方案将促进鲑鱼细胞系的功能遗传学研究，这些细胞系被广泛用作水产养殖传染病的模型系统。本文的在线版本 (10.1007/s10126-020-09995-y) 包含补充材料，可供授权用户使用。
Infectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide valuable models to study infectious diseases in salmonids, and genome editing using CRISPR/Cas systems provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas editing has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly used salmonid fish cell lines: Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90% cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture. The online version of this article (10.1007/s10126-020-09995-y) contains supplementary material, which is available to authorized users.
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