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

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. W...

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Detalles Bibliográficos
Autores principales: Huber, Anne, Dijkstra, Christine, Ernst, Matthias, Eissmann, Moritz F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9918790/
https://www.ncbi.nlm.nih.gov/pubmed/36853714
http://dx.doi.org/10.1016/j.xpro.2023.102076
Descripción
Sumario: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.