The creation of arrays of yeast strains each encoding a different protein with constant tags is a powerful method for understanding how genes and their proteins control cell function. As genetic tools become more sophisticated there is a need to create custom libraries encoding proteins fused with specialised tags to query gene function. These include protein tags that enable a multitude of added functionality, such as conditional degradation, fluorescent labelling, relocalization or activation and also DNA and RNA tags that enable barcoding of genes or their mRNA products. Tools for making new libraries or modifying existing ones are becoming available, but are often limited by the number of strains they can be realistically applied to or by the need for a particular starting library. We present a new recombination-based method, CATS – Cas9-Assisted Tag Switching, that switches tags in any existing library of yeast strains. This method employs the reprogrammable RNA guided nuclease, Cas9, to both introduce endogenous double strand breaks into the genome as well as liberating a linear DNA template molecule from a plasmid. It exploits the relatively high efficiency of homologous recombination in budding yeast compared with non-homologous end joining. The method takes less than 2 weeks, is cost effective and can simultaneously introduce multiple genetic changes, thus providing a rapid, genome-wide approach to genetic modification.

中文翻译：

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CATS：Cas9 辅助标签切换。一种在酵母中交换基因组肽标签的高通量方法


创建酵母菌株阵列，每个酵母菌株编码带有恒定标签的不同蛋白质，是了解基因及其蛋白质如何控制细胞功能的强大方法。随着遗传工具变得越来越复杂，需要创建编码与专门标签融合的蛋白质的定制库来查询基因功能。其中包括能够实现多种附加功能的蛋白质标签，例如条件降解、荧光标记、重新定位或激活，以及能够对基因或其 mRNA 产物进行条形码编码的 DNA 和 RNA 标签。用于创建新文库或修改现有文库的工具正在变得可用，但通常受到它们可以实际应用的菌株数量或特定起始文库的需求的限制。我们提出了一种新的基于重组的方法，CATS – Cas9 辅助标签转换，可以在任何现有的酵母菌株库中转换标签。该方法采用可重编程的 RNA 引导核酸酶 Cas9，将内源双链断裂引入基因组，并从质粒中释放线性 DNA 模板分子。与非同源末端连接相比，它利用了出芽酵母中同源重组相对较高的效率。该方法耗时不到两周，具有成本效益，并且可以同时引入多种遗传变化，从而提供了一种快速的、全基因组的遗传修饰方法。