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

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Abstract

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.

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Abbreviations

CFTR:

Cystic fibrosis transmembrane conductance regulator

CF:

Cystic fibrosis

ERAD:

ER-associated protein degradation

CRISPR/Cas:

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein

NHEJ:

Nonhomologous end joining

DSB:

Double-stranded break

PAM:

Protospacer adjacent motif

T7E1:

T7 endonuclease 1

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Acknowledgment

This work was supported by NRF-2011-002945, NRF-2011-0023701, and 2013R1A3A2042197 from the National Research Foundation (NRF), the Ministry of Science, ICT & Future Planning, Korea.

Author's contribution

WC acquired most of the data. JK design the whole study along with ML and JK analysed data. WC and JK wrote the manuscript. MS and HK helped the target selection and provided whole-in-one Cas9 vector and surrogate reporter plasmids. JP and WN measured the rescued ΔF508-CFTR function and JL provided the lentivirus expressing ΔF508-CFTR. All authors read and approved the final manuscript.

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Correspondence to Joo Young Kim.

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The authors declare that they have no conflicts of interest.

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Chung, W.Y., Song, M., Park, J. et al. Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing. Biotechnol Lett 38, 2023–2034 (2016). https://doi.org/10.1007/s10529-016-2190-4

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  • DOI: https://doi.org/10.1007/s10529-016-2190-4

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