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

Abstract

Summary: 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cancer
• CRISPR
• Organoids