One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids.

Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.

Intestinal organoid cocultures with microbes.

Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host-microbe interactions with great experimental control. This protocol comprises methods to coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.

Dawn of the organoid era: 3D tissue and organ cultures revolutionize the study of development, disease, and regeneration.

Novel and updated approaches of culturing cells in 3D are rapidly advancing our understanding of development, health, and disease. As tissues have been found to behave more realistically in 3D than in 2D cultures, organoid technology in combination with recent advances in the isolation and generation of stem cells, has rapidly become a promising concept in developmental and regenerative research. The development of all kinds of tissues can now be studied "in a dish," allowing more detailed observations of stem cell maintenance, morphogens, and differentiation. This review explores how organoids have revolutionized academic research over the last 4 decades, and how they may continue to do so. It also addresses remaining hurdles in 3D cell culturing, and how they may be overcome.