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

• Published in: STAR protocols Vol. 4; no. 1; p. 101978
• Main Authors: Chan, Dedrick Kok Hong, Collins, Scott David, Buczacki, Simon James Alexander
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.03.2023; Elsevier
• Subjects: CRISPR; Microscopy; Organoids; Protocol; Microscopy; Organoids; CRISPR
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101978

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. [Display omitted] •Derivation of adult stem-cell-derived human colonic organoids•Generation of target gene knockout using multi-guide RNA CRISPR-Cas9•Immunofluorescent validation of target gene knockout for organoids Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.