citations

                    Isogenic cell models of cystic fibrosis-causing variants in natively expressing pulmonary epithelial cells.
                

Authors:

Valley HC, Bukis KM, Bell A, Cheng Y, Wong E, Jordan NJ, Allaire NE, Sivachenko A, Liang F, Bihler H, Thomas PJ, Mahiou J, Mense M.

In:

Source: J Cyst Fibros
Publication Date: (2019)
Issue: 18(4): 476-483

Research Area:

Respiratory Research

Platform:

4D-Nucleofector® X-Unit

Experiment

The authors describe an adaptable CRISPR-based gene editing pipeline to efficiently and seamlessly (without antibiotic selection, additional mutations, or residual DNA elements such as loxP sites) create isogenic models of CF-causing variants in the genomic context of 16HBE14o- immortalized bronchial epithelial cells.

Cas9 RNP was prepared fresh for each experiment. 100 µM stock tracrRNA (or tracrRNA, ATTO550) and crRNAwere combined at equimolar concentrations for a final duplex concentration of 50 µM, incubated at 95 °C for 5 min, and then cooled to room temperature. RNP complex was prepared by mixing tracrRNA/crRNA duplex at a 1:1.2Mratio with Cas9 and incubating for 10–20 min at room temperature. 16HBE14ocells were nucleofected with the Lonza 4D-Nucleofector system using the Lonza SG cell line 4D X Kit S and program CM-137. Each reaction contained approximately 200,000 16HBE14o- cells in 20 µl of supplemented SG buffer, 4 µl of RNP complex, and 1 µl of 100 µM ssODN HDR template.

Abstract

Background: Assessment of approved drugs and developmental drug candidates for rare cystic fibrosis (CF)-causing variants of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) requires abundant material from relevant models.

Methods: Isogenic cell lines harboring CFTR variants in the native genomic context were created through the development and utilization of a footprint-less, CRISPR/Cas9 gene editing pipeline in 16HBE14o- immortalized bronchial epithelial cells.

Results: Isogenic, homozygous cell lines for three CFTR variants (F508del and the two most common CF-causing nonsense variants, G542X and W1282X) were established and characterized. The F508del model recapitulates the known molecular pathology and pharmacology. The two models of nonsense variants (G542X and W1282X) are sensitive to Nonsense Mediated mRNA Decay (NMD) and responsive to reference compounds that inhibit NMD and promote ribosomal readthrough.

Conclusions: We present a versatile, efficient gene editing pipeline that can be used to create CFTR variants in the native genomic context and the utilization of this pipeline to create homozygous cell models for the CF-causing variants F508del, G542X, and W1282X. The resulting cell lines provide a virtually unlimited source of material with specific pathogenic mutations that can be used in a variety of assays, including functional assays.

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