Data underlying chapter 5 of PhD thesis: Exploring the potential of yeast mitochondria for synthetic cell research

The attached datasets belongs to the PhD thesis of Charlotte Koster, more particularly Chapter 5 entitled "Exploration of mRNA-sized RNA import into <em>Saccharomyces cerevisiae</em> mitochondria by a combined synthetic biology and adaptive laboratory evolution approach".Abstract: Efficient gene integration using RNA-guided endonucleases has not yet been achieved in the mitochondrial genome. Import of nucleic acids into mitochondria, a controversial feature, is essential for implementation of Cas9-mediated genome engineering of mitochondria. Short RNA import naturally occurs in mitochondria, and several putative import mechanisms and determinants have been proposed. However to date, import of gene-length RNA, required for gene integration in the mitochondrial genome, has never been described. The goal of this study was to devise and test experimental strategies to detect and improve the import of mRNA-sized RNA in mitochondria, using S. cerevisiae as model. A first fluorescence-based screening approach, relying on mitochondrial import of a fluorescent protein encoding mRNA revealed weak and stochastic RNA import, independent of the import signal fused to the mRNA. This screening also suggested a positive impact of mitochondrial co-import of the mRuby2 fluorescent protein with the mRNA. An adaptive laboratory evolution (ALE) approach, imposing a strong selection pressure for mRNA import to mitochondria, was then designed and tested to improve mitochondrial mRNA import. While the ALE approach did not improve mitochondrial mRNA import in the present study, it is a promising, unambiguous method for future studies testing different RNAs or mutants.

本附带数据集源自夏洛特·科斯特（Charlotte Koster）的博士学位论文，具体对应其中第五章，题为《基于合成生物学与自适应实验室进化联用策略探究mRNA级RNA向酿酒酵母（Saccharomyces cerevisiae）线粒体的导入》。摘要：目前尚未实现在线粒体基因组中利用RNA引导的核酸内切酶完成高效基因整合。核酸向线粒体的导入这一颇具争议的研究方向，是实现Cas9介导的线粒体基因组工程不可或缺的必要前提。线粒体中天然存在短RNA的导入现象，目前已提出多种潜在的导入机制与决定因素。然而迄今为止，尚未有关于实现线粒体基因组基因整合所需的基因长度RNA导入的相关研究报道。本研究以酿酒酵母为模式生物，旨在设计并测试可用于检测并提升mRNA级RNA向线粒体导入效率的实验策略。首个基于荧光的筛选方案，通过检测编码荧光蛋白的mRNA向线粒体的导入过程，发现RNA导入效率偏弱且呈随机分布特征，且与mRNA所融合的导入信号无关。该筛选结果还表明，将mRuby2荧光蛋白与mRNA共同向线粒体导入，可产生积极影响。随后，研究人员设计并测试了一种自适应实验室进化（adaptive laboratory evolution, ALE）策略，通过对mRNA向线粒体的导入施加强选择压力，以提升线粒体mRNA的导入效率。尽管本研究中该ALE策略未能提升线粒体mRNA的导入效率，但该方法仍具备广阔应用前景，可为后续测试不同RNA或突变体的研究提供明确可靠的技术路径。