Project Summary Cell reprogramming represents a major advancement in biology, and has wide applications in regenerative medicine, disease modeling and drug screening. Somatic cells such as fibroblasts can be directly converted into induced neuronal (iN) cells via the forced expression of three transcription factors: Ascl1, Brn2 and Myt1l (BAM). However, a major challenge of cell reprogramming, especially iN reprogramming, is the low reprogramming efficiency, which has limited the translation of this technology for biomedical applications. Biophysical factors from the microenvironment have been shown to regulate many aspects of cell functions such as cell growth, migration and differentiation. Recently, we have shown that mechanical deformation of cell nucleus through microfluidic channels can promote open chromatin structure and enhance cell reprogramming, yet the underlying mechanisms are not well understood. Based on our recent findings, we hypothesize that a mechanopriming process such as mechanical squeezing of cell nucleus can induce NL reorganization and a permissive chromatin state to facilitate the activation of neuronal genes in the heterochromatin of fibroblasts, which promotes iN reprogramming and CRISPR- mediated gene editing/activation. To test our hypothesis, we propose three Specific Aims: (1) To investigate how nuclear deformation modulates the epigenetic state to enhance iN reprogramming; (2) To determine the role of nuclear lamina in mediating nuclear deformation-induced LAD dissociation, epigenetic changes and iN reprogramming; (3) To investigate the enhancement of CRISPR-mediated neuronal gene activation by mechanical squeezing. We have assembled a multidisciplinary team with expertise on cell engineering, microdevice fabrication, high-throughput genomic and epigenomic analysis, neuroscience, biosensors and CRISPR gene editing to work together and investigate the mechanical regulation of epigenetic state and cell reprogramming. We propose to optimize a high-throughput microfluidic device, further investigate the causative mechanisms and profile the genome-wide site-specific epigenetic changes induced by nuclear deformation, which can provide a rational basis for the design of site-specific gene editing for cell engineering. Accomplishment of this project will advance our understanding of how biophysical factors regulate cell reprogramming and the epigenetic state, and unravel new mechanisms of cell fate determination, which will have wide applications in gene editing, cell and tissue engineering, disease modeling and drug discovery.