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https://scholarbank.nus.edu.sg/handle/10635/245646
Title: | Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9 | Authors: | Chan, Dedrick Kok Hong Collins, Scott David Buczacki, Simon James Alexander |
Keywords: | Science & Technology Life Sciences & Biomedicine Biochemical Research Methods Biochemistry & Molecular Biology |
Issue Date: | 17-Mar-2023 | Publisher: | ELSEVIER | Citation: | Chan, Dedrick Kok Hong, Collins, Scott David, Buczacki, Simon James Alexander (2023-03-17). Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9. STAR PROTOCOLS 4 (1). ScholarBank@NUS Repository. | 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. | Source Title: | STAR PROTOCOLS | URI: | https://scholarbank.nus.edu.sg/handle/10635/245646 | ISSN: | 2666-1667 |
Appears in Collections: | Staff Publications Elements |
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Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-.pdf | Published version | 2.06 MB | Adobe PDF | OPEN | Published | View/Download |
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