Project summary Genetically engineered mouse models are an indispensable tool for studying human development and disease, enabling us to characterize gene functions in vivo, to investigate the cellular and molecular mechanisms of physiological and pathological processes, and to establish faithful disease models for drug screen. To date, most mouse models are engineered to recapitulate human disease phenotype, yet more sophisticated mouse models are in demand to study disease gene function in a native cellular background, to perform single cell based analyses and to set up high-throughput reporter-based screens for phenotypes or therapy. Fluorescent tagging of endogenous genes is a particularly powerful strategy, allowing the characterization of subcellular localization of proteins, identification of interaction partners, and isolation of specific cell populations in the context of development and disease. Engineering functional tags in an endogenous gene locus of interest preserves endogenous expression and minimizing genomic disruption, but the technology still remains inefficient, costly and laborious. We recently developed CRISRP-EZ (CRISPR RNP Electroporation of Zygotes), an electroporation-based technology that outperforms microinjection in efficiency, simplicity, cost, and throughput for mouse genome editing. Based on the CRISPR-EZ technology, we aim to develop novel technologies to achieve rapid fluorescent tagging of endogenous proteins with unprecedented efficiency, simplicity, throughput and cost saving. First, we will employ the self- complementing split-GFP system to achieve functional GFP tagging of endogenous genes in vivo using CRISPR-EZ. Second, we will develop CRISPR-READI (CRISPR RNP Electroporation and AAV Donor Infection) to engineer full length fluorescent protein tagging on endogenous loci in mice by AAV-mediated HDR editing. Taken together, our technologies will allow efficient and high throughput fluorescent tagging in mouse disease models for comprehensive mechanistic studies and powerful drug screening.
NIH ExPorter
NIH ExPorter