Strategies for genetic manipulation of adult stem cell-derived organoids

Organoid technology allows the expansion of primary epithelial cells from normal and diseased tissues, providing a unique model for human (patho)biology. In a three-dimensional environment, adult stem cells self-organize and differentiate to gain tissue-specific features. Accessibility to genetic manipulation enables the investigation of the molecular mechanisms underlying cell fate regulation, cell differentiation and cell interactions. In recent years, powerful methodologies using lentiviral transgenesis, CRISPR/Cas9 gene editing, and single-cell readouts have been developed to study gene function and carry out genetic screens in organoids. However, the multicellularity and dynamic nature of stem cell-derived organoids also present challenges for genetic experimentation. In this review, we focus on adult gastrointestinal organoids and summarize the state-of-the-art protocols for successful transgenesis. We provide an outlook on emerging genetic techniques that could further increase the applicability of organoids and enhance the potential of organoid-based techniques to deepen our understanding of gene function in tissue biology. Organ model: Making custom 3D models using genetic manipulation Advances in targeted genomic manipulation are expanding the utility of cultured organ models for studying disease in complex tissues. Mammalian cells can be coaxed to self-assemble and mature into organoids, structures that recapitulate key features and functions of various organs. Constantin Menche and Henner Farin from the Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany, have reviewed how technologies like the CRISPR/Cas9 genome editing system can be used to "customize" such organoid cultures. They highlight challenges associated with achieving efficient genetic manipulation in gastrointestinal organoid systems. These include delivering the necessary reagents for genome editing into cells, and ensuring that only the desired changes are made to the DNA. Under optimal conditions, researchers can test the tissue-level effects of deleting or altering genes, or study the impact of mutations that have been observed in patients.