TALE技术的优化及其在活细胞内成像的应用

TALE (Transcription Activator–Like Effectors) technology has become powerful imaging tool in visualizing of specific gene loci, in addition to widely used in targeted gene editing. However, there are still many defects of TALE in the application of targeted genome imaging, such as non-specific nucleolus-like aggregates, low signal to noise ratio. In order to resolve the problems of TALE technology, we intend to add resolvable short peptide at the end of the TALE, and further to increase the solubility of TALE and solve its defects in imaging. In detail, we plan to design, construct and screen TALE fusion peptide library (TALE-Linker-short peptide for solubilization-fluorescence protein), and obtain the optimized TALE fusion peptide. Furthermore, in order to validate the specificity and image quality of the optimized TALE fusion peptide, we plan to design kinds of TALE fusion peptides for targeting telomere, centromere, rDNA and the single gene loci MUC4, respectively, and observe the image quality by confocal microscopy. The successful construction of TALE platform provides a new method for revealing the functional relevance between spatial organization of chromatin and genome function, and will be further applied to study the changes of telomere length, centromere and rDNA in human aging cells.

TALE（转录激活因子样效应子）技术除广泛应用于靶向基因编辑外，也已成为强大的基因组成像工具，用于特定基因位点的可视化。然而，现有的TALE在基因组成像应用中存在诸多缺陷，如出现非特异性核仁样聚集体、信噪比低等。本项目拟采用在TALE末端添加促溶短肽的方法来增加TALE溶解度，进而解决其成像中的缺陷，具体是通过设计、构建并筛选特有的TALE融合肽库（TALE-Linker-促溶短肽-荧光蛋白），获得优选TALE融合肽。此外，为验证优选TALE融合肽对靶基因的特异性及成像效果，申请人拟分别设计靶向端粒、着丝粒、rDNA甚至单基因MUC4位点的TALE融合肽，并利用共聚焦显微镜成像技术进行成像观察与鉴定。TALE技术平台的成功搭建，将为揭示染色质空间组织和基因组功能之间的功能相关性提供新的方法，并可进一步应用于衰老细胞中端粒长短、着丝粒、rDNA等基因变化的研究中。

活细胞中特定基因组位点的可视化是研究细胞老化等多种生物过程中染色质结构动态变化的前提。然而，由于缺乏特异性高、信噪比高的荧光探针，限制了目前精确的基因组成像方法。我们发现，传统的转录激活因子样效应子（TALEs）倾向于形成蛋白质聚集体，从而影响其在成像应用中的性能。通过筛选，我们发现通过融合硫氧还蛋白与TALEs可以优化基于TALEs的基因组成像技术。使用硫氧还蛋白-融合的TALEs（TTALEs），我们实现了在不同的基因位点的高质量图像，并利用该技术在人类过早衰老模型中观察到端粒和着丝粒衰老相关（epi）基因组改变。重要的是，我们发现了核糖体DNA重复序列的损耗是人类衰老的一个分子标记。我们的研究建立了一种简单而稳健的用于精确监测染色质动态变化的成像方法。