Physiological Genomics

Project Description

Recent advances in genome engineering technologies have fundamentally transformed our capacity to manipulate genetic material with precision, opening up unprecedented avenues in basic research, biotechnology, and medicine. Among these tools, the CRISPR-Cas system has emerged as a particularly powerful and versatile platform for targeted genome editing and transcriptional regulation. Owing to its simplicity, programmability, and adaptability, CRISPR has seen widespread adoption across diverse genomic applications. Beyond genome editing, CRISPR has been successfully adapted for transcriptional activation (CRISPRa), enabling the upregulation of endogenous genes without altering the DNA sequence1. This strategy offers tremendous potential for the precise modulation of gene expression, functional genomic studies, and the engineering of desired cellular phenotypes in both therapeutic and biotechnological contexts2.

One particularly promising application of CRISPRa is in cell identity reprogramming — a strategy with transformative potential for regenerative medicine. By reactivating developmentally important genes, cell types lost to disease (e.g., neurons in neurodegenerative conditions) can potentially be replaced through in situ reprogramming of neighboring cells3,4. However, the clinical and in vivo application of CRISPR-based reprogramming has been significantly hindered by the limitations of viral delivery systems, which pose challenges in terms of delivery efficiency, immunogenicity, and potential genotoxicity.

To address this, we have developed a non-viral, RNP-based CRISPRa platform utilizing a potent transcriptional activator — dCas9-VPR5. We have successfully purified highly active dCas9-VPR protein from insect cells at high yield and demonstrated its efficient assembly with chemically synthesized guide RNAs into functional dCas9-VPR ribonucleoprotein complexes (dRNPs). These complexes can be delivered with high efficiency into human stem cells, their differentiated progeny, and primary cells. Our data show that targeted gene activation via dRNPs is both rapid and transient, with induction levels reaching up to 100000-fold, including for developmentally silenced genes. Optimization of dosing parameters enables the simultaneous activation of as many as 35 genes, underscoring the scalability of the system for complex reprogramming applications.

Most recently, we demonstrated that dRNPs can drive cell fate specification and conversion in vitro — for instance, inducing neuronal differentiation from human stem and progenitor cells⁵. As a next step, we aim to extend this technology to in vivo settings. To facilitate this, we have generated novel mouse reporter lines capable of visualizing CRISPRa-induced gene activation and cell reprogramming (unpublished). By combining these models with optimized strategies for RNP delivery, our goals are to:

• Verify, characterize, and quantify CRISPRa RNP delivery and gene activation in vivo;
• Establish functional reprogramming protocols in vivo, particularly within neurodegenerative and metabolically compromised environments.

This project has the potential to establish a safe, efficient, and scalable framework for non-viral, CRISPRa-mediated cell therapy, paving the way for in situ regeneration strategies in a range of disease contexts.