Differentiation and CRISPR-Cas9-mediated genetic engineering of human intestinal organoids

Adriana Martinez-Silgado, Fjodor A. Yousef Yengej, Jens Puschhof, Veerle Geurts, Charelle Boot, Maarten H. Geurts, Maarten B. Rookmaaker, Marianne C. Verhaar, Joep Beumer, and Hans Clevers

Published: 16 September 2022


Intestinal organoids are three-dimensional cultures that resemble key aspects of the epithelium of origin. Here, we describe how to differentiate human small intestinal organoids by combining growth media variations and genetic engineering. We detail the differentiation of human intestinal organoids in the presence and absence of BMP agonists to recapitulate a broader scope of functional cell states found in vivo. Using transient overexpression of the transcription factor Neurogenin-3, we describe the enhancement of differentiation toward rare enteroendocrine cells.

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