Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia.
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia.
ACS Synth Biol. 2021 Feb 19;10(2):297-308. doi: 10.1021/acssynbio.0c00426. Epub 2021 Jan 27.
The marine yeast is of high importance in the food, chemical, and medical industries. is also a popular model for studying molecular mechanisms of halo- and osmotolerance. The absence of genome editing technologies hampers research and limits its biotechnological application. We developed novel and efficient single- and dual-guide CRISPR systems for markerless genome editing of . The single-guide system allows high-efficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno- and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of . The developed CRISPR systems allow efficient genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.
海洋酵母在食品、化学和医疗行业具有重要地位。它也是研究耐盐和耐渗透压的分子机制的常用模式生物。缺乏基因组编辑技术限制了的研究和其生物技术应用。我们开发了新型高效的单引导和双引导 CRISPR 系统，用于无标记基因组编辑。单引导系统可实现高达 95%的基因或调控元件的高效突变。双引导系统适用于基因组位点的高效缺失。我们使用这些工具研究了 26S 蛋白酶体的转录调控，26S 蛋白酶体是一种依赖于 ATP 的蛋白酶复合物，其正常功能对所有细胞和生物体都是至关重要的。我们开发了一种遗传方法来通过调节其必需亚基来控制 26S 蛋白酶体的活性。突变株对基因毒性和蛋白毒性应激以及高盐度和高渗透压敏感，这表明蛋白酶体对的极端适应特性有一定贡献。开发的 CRISPR 系统允许高效的基因组工程，为控制蛋白酶体活性提供了一种遗传途径，应推进该酵母的应用。
