Abstract

The identification of LGR5+ intestinal stem cells helped us to understand various aspects of adult stem cells and led to the establishment of primary 3D intestinal organoid culture system from mouse and human tissues. This novel culture system faithfully recapitulates various aspects of the intestinal epithelium in vitro with remarkable long-term expansion capacity and genetic stability. Thus, the model is recognised as a suitable in vitro model system for genetic studies. To exploit all the potential of this culture, protocols have been fully optimised for primary establishment, maintenance, cryopreservation, plasmid transfection and viral transduction. A number of examples will be shown to introduce how to apply CRISPR technology and organoid models for genetic studies, including simultaneous paralogue knockout, functional genetic screening and precise gene correction.