基于超分辨FLIM方法的活细胞基因编辑动态检测

New gene editing techniques represented by CRISPR-Cas9 can efficiently recognize and edit the target genes in living cells, is of great scientific significance and economic value in the fields of gene therapy, genetic improvements of species and so on. However, there is a lack of established optical techniques for real-time dynamic analysis of gene editing process in living cells in a high-resolution (nano-scale) and quantitative way. Here, this project provides a multimodal super-resolution stimulated emission depletion (STED) microscopy combined with a two-photon excited fluorescence lifetime imaging microscopy (FLIM) optical technique for monitoring gene editing. STED technique can provide a resolution of ~50-100 nm of fluorescent dye labeled DNA. Meanwhile, FLIM modality will be used to analyze the activities of Cas9 enzymes as well as to monitor the interaction between enzymes and labeled DNA strands within 1-10 nm, through the fluorescence resonance energy transfer (FRET) method. As a result, STED and FLIM-FRET data sets will be mutually integrated to improve the resolution capabilities of nanoscopic imaging of bio-molecules and to monitor micro-environmental changes in the area of gene editing. Moreover, the development of this STED/FLIM super-resolution imaging method can provide a fast and efficient detection method for further optimization of gene editing efficiency. In conclusion, this project will establish a new optical STED/FLIM multimodal optical platform for quantitative analysis of gene editing process in live cells. The establishment of this method has important significance and application value in the field of gene editing inside living cells, which is also highly demanded in a broad area of quantitative analysis of life sciences.

以CRISPR-Cas9为代表的新型基因编辑技术可在活细胞中特异性识别及编辑基因，在基因治疗、物种改良等方面具有重大意义和经济价值，但目前缺乏对活细胞中基因编辑过程进行纳米分辨和定量表征的合适的实时动态光学方法。本项目提出并发展一种受激发射损耗（STED）显微成像和双光子激发荧光寿命显微成像（FLIM）相结合的基因编辑多模态光学表征方法，利用STED方法获得荧光标记靶DNA超分辨结构信息，利用FLIM-FRET（荧光共振能量转移）方法获得Cas9等内切酶活性信息。两种成像方法联用，可实现活细胞基因编辑过程中核酸内切酶作用区域的生物分子和微环境变化的动态监测，从而为基因编辑效率的优化提供一种快速、高效的检测方法。本项目将建立一种对基因编辑进行活细胞定量分析的STED/FLIM多模态光学方法，在活细胞基因编辑研究领域具有重要科学意义和应用价值，并有望在生命科学定量分析领域获得广泛应用。

细胞是生命活动的基本单位，目前细胞生物学中一个研究热点是以CRISPR-Cas9为代表的新型基因编辑技术，其可在活细胞中特异性识别及编辑基因，在基因治疗、物种改良等方面具有重大意义和经济价值，但目前缺乏对活细胞中基因编辑过程进行纳米分辨和定量表征的合适的实时动态光学方法。针对该问题，本项目提出并发展一种受激发射损耗（STED）显微成像和荧光寿命显微成像（FLIM）相结合的多模态光学表征方法，用来检测基因编辑过程，具体包括：在已有的基础上，提出并发展了一种对活细胞内特定区域实现STED/FLIM多模态成像方法；进一步对活细胞内生命过程过程中的关键分子开展光学显微成像。.与传统的生化检测及细胞生物学研究方法相比，本方法具有实时动态、无损、灵敏度高、分辨率好等优点，达到了本项目预期的单细胞细胞核内特定区域的定量检测等应用目标，并为研究活细胞的细胞核内染色质动态过程提供了一种通用的定量分析的STED/FLIM多模态光学方法，有望在细胞生物学机制研究特别是活细胞中功能研究及基因活性监测得到广泛应用。.综上所述，本项目具有较好的研究意义和应用价值。本项目研究成果发表期刊论文21篇（SCI收录20篇、EI收录1篇）及EI收录的会议论文1篇；申请国家发明专利2项，申请并获授权实用新型专利3项；参加学术会议4次；培养博士后2名、博士研究生1名、硕士研究生4名。