Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9
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 col...
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Published in | STAR protocols Vol. 4; no. 1; p. 101978 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
17.03.2023
Elsevier |
Subjects | |
Online Access | Get full text |
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Summary: | 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.
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•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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Technical contact Lead contact |
ISSN: | 2666-1667 2666-1667 |
DOI: | 10.1016/j.xpro.2022.101978 |