Soc media

A lentiviral vector containing Cas9 and a human U6 promoter for sgRNA expression (LentiCRISPRv2: Addgene 52961) was digested with BsmBI (NEB R0580) for 3 h at 55 °C. The digested vector was then purified using a Qiaquick PCR purification column (Qiagen 28104). Gibson Assembly reactions containing 200 ng of digested vector, 36 ng of insert (containing pooled library), and 10 μL of Gibson Assembly Master Mix (NEB E2611S) were then incubated at 50 °C for 1 h, and subsequently transformed into 200μL of Stbl4 electrocompetent bacteria (Thermo 11635018). Transformed cells were resuspended in 8 mL of SOC media (Invitrogen 15544034) and allowed to recover for 1 h shaking before being used to inoculate 150 mL of LB media supplemented with carbenicillin. After 16 h of further growth, plasmid DNA containing the sgRNA library was isolated via a Qiagen Plasmid Plus MaxiPrep kit (Qiagen 12963).

One Shot MAX Efficiency DH5α-T1^R competent cells (Invitrogen, Massachusetts, USA) were incubated with 1 µl of plasmid on ice for 30 min. These cells were then heat shocked at 42 °C for 30 s, returned to ice for 2 min, and grown in 950 µl SOC media (Invitrogen, Massachusetts, USA) at 37 °C with shaking for 1 h. Resulting transformed Escherichia coli cells were plated on LB agar with ampicillin (100 µg/ml; Sigma Aldrich, Missouri, USA) and grown over night at 37 °C. Colonies were then grown 8 h in 10 ml LB medium with ampicillin (100 µg/ml) followed by growing 400 ml to extract plasmids. Transfection-grade plasmid DNA was obtained using the NucleoBond Xtra Midi or Maxi kits (Macherey–Nagel, Düren, Germany). Final DNA product was assessed for quantity and quality using NanoDrop 200- (Thermo Fisher Scientific, Massachusetts, USA).

CFTR variants were generated using the SPRINP mutagenesis method (SI Appendix, Table S2) (54 (link)). Briefly, mutagenic primers were designed to be complementary to the template plasmid except for the mutated bases and to be 15 to 45 nucleotides in length. Plasmid containing CFTR cDNA was amplified in separate reactions containing forward or reverse primer. The single-primer products of these reactions were combined and denatured at 95 °C for 5 min and gradually cooled to 37 °C over the next 5 min. The sample was then digested by DpnI for 4 h. Then, 5 µL of sample was added to 50 µL of competent XL2Blue cells for transformation and incubated on ice for 30 min. The bacteria were then heat-shocked at 42 °C for 45 s and allowed to recover on ice for 2 min. Following that, 200 µL of warmed SOC media (Invitrogen) was then added directly to the cells, and the mixture was allowed to shake at 225 RPM in a 37 °C incubator for 30 min. Then, 200 µL of this mixture was spread on LB/ampicillin plates and left to incubate at 37 °C overnight. Random colonies were then picked and expanded in LB/ampicillin. Plasmid DNA was then purified (QIAGEN Plasmid Kit) and sequenced (Genewiz).