Department of Biology, Centre for Applied Synthetic Biology, Concordia University, Montréal, QC H4B1R6, Canada.
G3 (Bethesda). 2022 Jul 29;12(8). doi: 10.1093/g3journal/jkac154.
The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR-associated protein 9 (Cas9)-based genome editing tools, we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 toolkit with 3 components: (1) a set of optimized Streptococcus pyogenes Cas9-guide RNA expression vectors with 5 selectable markers and the option to either preclone or cotransform the gRNAs; (2) vectors for the one-step construction of integration cassettes expressing an untagged or green fluorescent protein/red fluorescent protein/hemagglutinin-tagged gene of interest at one of 3 levels, supporting localization and overexpression studies; and (3) integration cassettes containing moderately expressed green fluorescent protein- or red fluorescent protein-tagged compartmental markers for colocalization experiments. These components allow rapid, high-efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. To demonstrate the ease of use, we applied our complete set of compartmental markers to colabel all target subcellular compartments with green fluorescent protein and red fluorescent protein. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle that allows the stable genomic integration of DNA with the ease of use approaching that of transforming plasmids.
酵母酿酒酵母的遗传可操作性使其成为基础研究的重要模型生物和代谢工程的目标。为了简化标记基因和隔室标记的引入，使用强大的规律成簇间隔短回文重复序列 (CRISPR) - CRISPR 相关蛋白 9 (Cas9) 为基础的基因组编辑工具，我们构建了一个无标记酵母定位和过表达 (MyLO) CRISPR-Cas9 工具包，具有 3 个组件：(1) 一组优化的酿脓链球菌 Cas9 向导 RNA 表达载体，带有 5 个可选择标记，并且可以预克隆或共转化 gRNA；(2) 用于一步构建整合盒的载体，在 3 个水平之一表达未标记或绿色荧光蛋白/红色荧光蛋白/血凝素标记的目的基因，支持定位和过表达研究；(3) 整合盒包含中度表达的绿色荧光蛋白或红色荧光蛋白标记的隔室标记物，用于共定位实验。这些组件允许快速、高效的基因组整合和修饰，仅需瞬时选择 Cas9 载体，从而实现无标记转化。为了展示其易用性，我们应用了整套隔室标记物，用绿色荧光蛋白和红色荧光蛋白对所有目标亚细胞隔室进行共标记。因此，MyLO 工具包将 CRISPR-Cas9 技术包装成一个灵活、优化的套件，允许 DNA 的稳定基因组整合，使用起来就像转化质粒一样简单。
