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.
