CAREER: Investigating Chromatin Dynamics Underlying Activity-Induced Neuronal Transcription Using CRISPR Technologies

哺乳动物基因组在细胞核中折叠成三维（3D）结构。长达2米、含有30亿个碱基对（bp）的灵长类动物DNA是如何融入10微米的细胞核中的，几十年来一直吸引着生物学家。显微成像和DNA测序方法揭示了DNA领土和结构域的不同结构单元，当细胞改变命运或处于环境压力下时，这些结构单元会动态变化。这些DNA结构域如何在功能上影响基因表达和细胞活性仍不清楚。为了解决这一重要问题，该项目旨在开发一种新的基于CRISPR的技术平台，以揭示3D基因组组织和基因表达之间的因果关系。该项目将开发一个在线教育平台，由实践课程模块组成，旨在激发本科生和高中生对CRISPR基因编辑的广泛兴趣。来自不同文化或社会经济背景的学生将通过远程学习平台接触尖端的基因组编辑工具，知识和技术。预计这些教育活动将吸引STEM学生和教师，促进代表性不足的群体的参与，并向公众宣传和教育新型基因编辑生物技术的重要性和负责任的使用。该项目将培养基因组编辑和基因组学领域的研究生，以促进科学交流、教育和研究。该项目的研究目标是开发一种基于CRISPR的新技术平台，以阐明3D染色质相互作用的动力学和时间尺度及其与基因转录的因果关系。PI实验室的初步工作已经开发了几种基于CRISPR的方法，例如用于活细胞成像的LiveFISH，用于转录扰动的CRISPRa/i，以及用于基因组位点的诱导和可逆重新定位的CRISPR-GO。该项目将开发一个集成平台，能够在单个活神经元细胞的纵向时间尺度上对任何DNA/RNA/蛋白质进行超分辨率水平的同时扰动和多色成像。以诱导的人类神经元为模型系统，具体的科学目标是：1）通过活细胞成像测量单个神经元中信号刺激基因位点处的染色质成环的时间尺度; 2）通过多色超分辨率成像研究神经元激活后成环、mRNA转录和蛋白质翻译的动力学; 3）通过用CRISPRa/i靶向干扰基因转录或用CRISPR-GO靶向干扰成环来确定成环与转录之间的相互关系。该项目的成功完成将揭示染色质环形成和复杂性的新知识，并揭示它们与转录和细胞活性的因果功能关系。这项研究获得的见解有望形成生物技术基础，可用于操纵和设计未来功能基因组学研究的3D基因组。该项目由MCB部门的系统与合成生物学和遗传机制集群共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Mammalian genomes are folded into 3-dimensional (3D) structures in the nucleus. How the 2-meter-long primate DNA with 3-billion base pairs (bp) fit into a 10-micrometer nucleus has intrigued biologists for decades. Microscopy imaging and DNA sequencing methods have revealed distinct structural units of DNA territories and domains, which change dynamically when cells change fate or are under environmental stress. How these DNA structural domains functionally impact gene expression and cellular activity remains unclear. To address this important question, the project aims to develop a new CRISPR-based technology platform to reveal the causal relationship between the 3D genome organization and gene expression. The project will develop an online education platform consisting of hands-on curricula modules designed to stimulate broad interest on CRISPR gene editing among undergraduates and high school students. Students from diverse cultural or socio-economic backgrounds will gain exposure to cutting-edge genome editing tools, knowledge and techniques via the remote learning platform. These educational activities are expected to engage STEM students and teachers, promote participation of underrepresented groups, and inform and educate the public about the importance and responsible use of novel gene editing biotechnologies. The project will train graduate students in the field of genome editing and genomics for science communication, education and research.The research objective of the project is to develop a novel CRISPR-based technology platform to elucidate dynamics and timescales of 3D chromatin interactions and their causal relationship to gene transcription. Preliminary work in the PIs lab has developed several CRISPR-based methods such as LiveFISH for live cell imaging, CRISPRa/i for transcription perturbation, and CRISPR-GO for inducible and reversable repositioning of genomic loci. The project will develop an integrated platform enabling simultaneous perturbation and multi-color imaging of any DNA/RNA/protein on super-resolution level over longitudinal timescales in single living neuron cells. Using induced human neurons as a model system, the specific scientific goals are to: 1) measure the timescales of chromatin looping at signal-stimulated gene loci via live-cell imaging in single neurons; 2) study dynamics of loop formation, mRNA transcription and protein translation via multiple-color super-resolution imaging after neuronal activation; 3) determine the mutual relationship between looping and transcription by targeted perturbation of gene transcription with CRISPRa/i or targeted perturbation of looping with CRISPR-GO. Successful completion of the project will reveal new knowledge of the chromatin loop formation and complexity and uncover their causal functional relationship to transcription and cell activity. The insights gained by this research are expected to form the biotechnology basis that can be used to manipulate and engineer the 3D genome for future functional genomics research. The project is jointly funded by the Systems and Synthetic Biology and Genetic Mechanisms clusters of the MCB Division.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.