Abstract：
Most CRISPR/Cas9 applications in yeast rely on a plasmid-based expression of Cas9 and its guide RNA (gRNA) containing a 20-nucleotides (nts) spacer tailored to each genomic target. The lengthy assembly of this customized gRNA requires at least 3-5 days for its precloning in Escherichia coli, purification, validation, and cotransformation with Cas9 into a yeast strain. Here, we constructed a series of 12 EasyGuide plasmids to simplify CRISPR/Cas9 applications in Saccharomyces cerevisiae. The new vectors provide templates for generating PCR fragments that can assemble up to six functional gRNAs directly into yeasts via homologous recombination between the 20-nts spacers. By dispensing precloning in E. coli, yeast in vivo gRNA assembly significantly reduces the CRISPR/Cas9 experimental workload. A highly efficient yeast genome editing procedure, involving PCR amplification of gRNAs and donors, followed by their transformation into a Cas9-expressing strain, can be easily accomplished through a quick protocol.
摘要：
酵母中的大多数CRISPR/Cas9应用依赖于Cas9及其包含针对每个基因组靶标定制的20个核苷酸(nts)间隔区的指导RNA(gRNA)的基于质粒的表达。这种定制的gRNA的长时间组装需要至少3-5天的时间才能在大肠杆菌中预克隆，净化,验证,并与Cas9共转化为酵母菌株。这里,我们构建了一系列12个EasyGuide质粒，以简化CRISPR/Cas9在酿酒酵母中的应用。新载体提供了用于产生PCR片段的模板,所述PCR片段可以通过20-nts间隔区之间的同源重组将多达6个功能性gRNA直接组装到酵母中。通过在大肠杆菌中分配预克隆，酵母体内gRNA组装显著降低CRISPR/Cas9实验工作量。一种高效的酵母基因组编辑程序，涉及gRNA和供体的PCR扩增，然后转化为Cas9表达菌株，可以通过快速协议轻松完成。
