Protein tagging is an effective method for characterizing a gene of interest. Tagging can be accomplished in vivo in Saccharomyces cerevisiae by chromosomal integration of a PCR-amplified cassette. However, common tagging cassettes are not suitable for in situ N-terminal tagging when we aim to preserve the gene's endogenous promoter. Existing methods require either two rounds of homologous recombination or a relatively complex cloning process to construct strains with N-terminal protein tags. Here, we describe a simple CRISPR/Cas9-based method for seamless N-terminal tagging of yeast genes that preserves their endogenous promoter. This method enables the generation of N-terminally tagged strains by introducing an expression vector containing the cas9 gene and a specific gRNA for cleaving the 5′ end of the target gene's protein-coding sequence, along with donor DNA containing the tag sequence and homology arms. gRNA cloning was executed by inverse PCR instead of the conventional method. After verifying the tag, the Cas9 and gRNA expression plasmids were eliminated without using antibiotic-containing medium. By this method, we generated strains that express N-terminally tagged subunits of the TORC1 protein kinase complex and found that these strains are comparable to strains made by conventional methods. Thus, our method provides a cost-effective alternative for seamless N-terminal tagging in baker's yeast.

中文翻译：

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基于 CRISPR/Cas9 的酿酒酵母 N 端蛋白无缝标记方法


蛋白质标签是表征感兴趣基因的有效方法。通过 PCR 扩增盒的染色体整合，可以在酿酒酵母体内完成标记。然而，当我们的目标是保留基因的内源启动子时，常见的标记盒不适合原位 N 末端标记。现有方法需要两轮同源重组或相对复杂的克隆过程来构建带有N端蛋白质标签的菌株。在这里，我们描述了一种基于 CRISPR/Cas9 的简单方法，用于对酵母基因进行无缝 N 末端标记，从而保留其内源启动子。该方法通过引入包含cas9基因和用于切割靶基因蛋白质编码序列 5' 端的特定 gRNA 的表达载体，以及包含标签序列和同源臂的供体 DNA，从而能够生成 N 末端标记菌株。 gRNA克隆是通过反向PCR而不是常规方法进行的。验证标签后，无需使用含抗生素培养基即可消除 Cas9 和 gRNA 表达质粒。通过这种方法，我们生成了表达 TORC1 蛋白激酶复合物 N 末端标记亚基的菌株，并发现这些菌株与传统方法制备的菌株相当。因此，我们的方法为面包酵母中的无缝 N 末端标记提供了一种经济高效的替代方案。