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

Dedrick Kok Hong Chan; Scott David Collins; Simon James Alexander Buczacki

doi:10.1016/j.xpro.2022.101978

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

STAR Protoc. 2023 Mar 17;4(1):101978. doi: 10.1016/j.xpro.2022.101978. Epub 2023 Jan 3.

Authors

Dedrick Kok Hong Chan¹, Scott David Collins¹, Simon James Alexander Buczacki²

Affiliations

¹ Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK.
² Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK. Electronic address: [email protected].

Abstract

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.

Keywords: CRISPR; Microscopy; Organoids.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adult
CRISPR-Cas Systems* / genetics
Coloring Agents
Gene Editing* / methods
Gene Knockout Techniques
Humans
Organoids
RNA / genetics

Substances

Coloring Agents
RNA

Grants and funding

C14094/A27178/CRUK_/Cancer Research UK/United Kingdom