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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使用核糖核蛋白复合物对多种鲑鱼细胞系进行高效基因组编辑
DOI：
10.1101/2020.04.03.022038
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Gratacap R
中科院分区：
文献类型：
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作者：
Gratacap R
传染病和寄生虫病对鲑科鱼类水产养殖的经济和动物福利产生重大负面影响。更好地了解宿主对这些疾病的反应和遗传抗性的功能基础是制定预防和治疗方案的关键。细胞系为研究鲑鱼感染性疾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一条令人兴奋的途径。虽然CRISPR/Cas编辑已经在奇努克鲑鱼细胞系（CHSE-214）中成功地进行，但迄今为止还没有报道编辑源自最商业相关的鲑鱼物种大西洋鲑鱼和虹鳟鱼的细胞系，这些鲑鱼物种难以扩增，因此使用慢病毒介导的方法进行编辑。在目前的研究中，使用核糖核蛋白（RNP）复合物对鲑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Cas 12 a的RNP的电穿孔在所有测试系的靶向编辑（通常> 90%的细胞被编辑）方面是有效的，并且酶的选择扩大了这些物种的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，鲑鱼细胞系被广泛用作水产养殖中传染病的模型系统。
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.