While readily achieved in cell lines, the application of CRISPR-Cas9 gene editing in human-derived organoids suffers from limited efficacy and complex protocols. Here, we describe a multi-guide RNA CRISPR-Cas9 gene-editing protocol which efficiently achieves complete gene knockout in adult human colonic organoids. This protocol also describes crucial steps including how to harvest patient tissue to maximize gene-editing efficacy and a technique to validate gene knockout following editing with immunofluorescent staining of the organoids against the target protein.
Journal article
2023-03-17T00:00:00+00:00
4
CRISPR, Microscopy, Organoids, Humans, Adult, Gene Knockout Techniques, CRISPR-Cas Systems, Gene Editing, Organoids, Coloring Agents, RNA