Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9

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.

Automated summary

In this study, a multi-guide RNA CRISPR-Cas9 gene-editing protocol is described that enables efficient complete gene knockout in adult human colonic organoids. The protocol also details crucial steps for maximizing gene-editing efficacy through tissue harvesting and validating gene knockout using immunofluorescent staining of the organoids against the target protein.