Generation of gene-of-interest knockouts in murine organoids using CRISPR-Cas9

Anne Huber; Christine Dijkstra; Matthias Ernst; Moritz F Eissmann

doi:10.1016/j.xpro.2023.102076

Generation of gene-of-interest knockouts in murine organoids using CRISPR-Cas9

STAR Protoc. 2023 Mar 17;4(1):102076. doi: 10.1016/j.xpro.2023.102076. Epub 2023 Jan 30.

Authors

Anne Huber¹, Christine Dijkstra², Matthias Ernst², Moritz F Eissmann³

Affiliations

¹ Olivia Newton-John Cancer Research Institute and La Trobe School of Cancer Medicine, La Trobe University, Melbourne, VIC 3084, Australia. Electronic address: [email protected].
² Olivia Newton-John Cancer Research Institute and La Trobe School of Cancer Medicine, La Trobe University, Melbourne, VIC 3084, Australia.
³ Olivia Newton-John Cancer Research Institute and La Trobe School of Cancer Medicine, La Trobe University, Melbourne, VIC 3084, Australia. Electronic address: [email protected].

Abstract

Gene-of-interest knockout organoids present a powerful and versatile research tool to study a gene's effects on many biological and pathological processes. Here, we present a straightforward and broadly applicable protocol to generate gene knockouts in mouse organoids using CRISPR-Cas9 technology. We describe the processes of transient transfecting organoids with pre-assembled CRISPR-Cas9 ribonucleoprotein complexes, organoid cell sorting, and establishing clonal organoid culture pairs. We then detail how to confirm the knockout via Western blot analysis.

Keywords: CRISPR; Cancer; Organoids.

Publication types

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

MeSH terms

Animals
Blotting, Western
CRISPR-Cas Systems* / genetics
Clone Cells
Gene Knockout Techniques
Mice
Organoids*