Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

中文翻译：

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使用新的整合位点扩展酿酒酵母基因组的 EasyClone-MarkerFree 工具包


生物技术生产需要遗传稳定的重组菌株。为了确保基因组稳定性，重组DNA通常被整合到宿主菌株的基因组中。已经开发出多种遗传工具用于将基因组整合到面包酵母酿酒酵母中。此前，我们开发了一个载体工具包 EasyClone-MarkerFree，用于稳定整合到酿酒酵母 X、XI 和 XII 染色体上的 11 个位点。无标记整合是通过 CRISPR-Cas9 系统实现的。在这项研究中，我们通过位于不同染色体上的八个额外基因间整合位点扩展了试剂盒。新站点整合效率达到80%以上。所有八个位点的绿色荧光蛋白 (gfp) 表达水平与原始 EasyClone-MarkerFree 工具包中的 XI-2 位点相似或更高。细胞生长没有受到整合到新八个地点中任何一个的影响。八载体扩展试剂盒可从 AddGene 获得。