citations

                    Highly efficient genome editing in primary bronchial epithelial cells establishes FOXJ1 as essential for ciliation in human airways
                

Authors:

Radu Rapiteanu??1, Tina Karagyozova1, Natalie Zimmermann2, Gareth Wayne1, MatteoMartufi1, Nikolai N Belyaev2, Kuljit Singh1, Joanna Betts1, Soren Beinke2 and Klio Maratou1

In:

Source: BioResearch Open Access
Publication Date: (2019)
Issue: 01: 01

Research Area:

Cancer Research/Cell Biology

Respiratory Research

Cells used in publication:

Epithelial, bronchial (NHBE), human: Species: human; Tissue Origin: lung
Epithelial, bronchial (DHBE), human COPD: Species: human; Tissue Origin: lung

Culture Media:

Bronchial Epithelial Cell Growth Medium

Platform:

4D-Nucleofector® X-Unit

Experiment

Primary cell culture experiments: Basal HBECs were cultured in antibiotic-free BEGM media (Lonza, CC-3170). Untransfected and genome edited basal cells were trypsinized (Lonza, CC-5034), spun down for 4min at 180xg and 200,000 cells were resuspended in 200µl BEGM and seeded in individual, collagen coated, apical wells of HTS transwell plates (Corning, 3378). 1ml of BEGM media
was added to the basolateral well. After 48h, media was aspirated from the apical well to create the ALI and the transwells were transferred to 24 well plates containing 1ml fresh differentiation media (either 3D or Pneumacult media). 3D media was used throughout this study, expect where clearly specified otherwise. 3D media was made up as a 1:1 mixture of SABM (Lonza, CC-3119) and DMEM (Gibco, 21969-035) to which SAGM supplements (Lonza, CC-4124) were added, omitting retinoic acid. Retinoic acid was added fresh, prior to media changes. Both Pneumacult and 3D media were kept antibiotic free. PneumaCult-ALI Medium was made up as per manufacturer’s recommendations.

Genome editing with Nucleofection: tracrRNA and crRNA were resuspended in Duplex Buffer (IDT, 11-01-03-01) to 100µM and annealed to a final concentration of 50µM. The mixture was heated to 90°C for 3min and cooled down at RT. 90pmoles of AltR S.p. Cas9 were assembled with 450pmoles crRNAtracrRNA hybrid (molar ratio of gRNA : Cas9 = 5:1) resulting in a 10.5µl volume solution to which 0.9µl of electroporation enhancer (100µM; IDT, 1075916) were added. This complex is stable and was left at RT while cells were prepared. HBEC were thawed and briefly expanded as described above. Upon reaching 70% confluency, basal HBECs were washed in HBSS (Lonza, CC-5034), trypsinized (Lonza, CC-5034) and 200.000 cells were resuspended in 8.6µl P3 Primary Cell Nucleofector Solution (Lonza, V4XP-3032). The cell solution was then mixed with the RNP solution, transferred to a well of a 16-well Nucleocuvette Strip (Lonza, V4XP-3032) and electroporated using the CM-113 program on the Amaxa 4D Nucleofector (Lonza). Electroporated cells were then swiftly transferred to 6 well plates containing 2mL pre-warmed BEGM and incubated at 37°C.

Abstract

The structure and composition of the bronchial epithelium is altered in respiratory diseases such as COPD and asthma, in which goblet cell hyperplasia and reduced numbers of ciliated cells impair mucociliary clearance. We describe a robust genome editing pipeline to interrogate modulators of primary human bronchial epithelial cell (HBEC) differentiation and function. By employing plasmid- and virus-free delivery of CRISPR/Cas9 to human airway basal cells we achieve highly efficient gene inactivation without the need for positive selection. Genome edited cells are differentiated at air liquid interface (ALI) into a pseudostratified epithelium. We focus on profiling ciliation using imaging cytometry coupled to confocal microscopy and immunohistochemistry. To our knowledge, this is the first study to describe highly efficient genome editing of ALI cultured primary HBECs. As proof of concept, we establish that inactivation of the gene encoding the transcription factor FOXJ1 in primary human airway basal cells precludes ciliation in ALI differentiated bronchial epithelia.