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.