Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing

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• 作者：Woo Young Chung ; Myungjae Song ; Jinhong Park ; Wan Namkung…
• 关键词：CRISPR/Cas9 ; ΔF508 ; CFTR ; Epithelial cell ; Genome editing ; T84 cell line
• 刊名：Biotechnology Letters
• 出版年：2016
• 出版时间：December 2016
• 年：2016
• 卷：38
• 期：12
• 页码：2023-2034
• 全文大小：2,505 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Microbiology
  Biotechnology
  Applied Microbiology
  Biochemistry
  
• 出版者：Springer Netherlands
• ISSN：1573-6776
• 卷排序：38

文摘

Objectives: To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. Results: CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. Conclusions: This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.