title: Deep Mutationa Scan CFTR Variants
design: A pcDNA5 vector, in which we inserted CFTR, a triple hemagglutinin (HA) tag in the fourth extra-cellular loop, an internal ribosome entry site- eGFP cassette, and a Bxb1 recombination site was used to generate a molecular library of barcoded CF variants. We first installed a randomized ten nucleotide unique molecular identified (UMI) upstream of the Bxb1 recombination site using nicking mutagenesis in place of the CMV promoter. A plasmid preparation containing WT plasmids with scrambled UMIs was used as a template for 106 individual site-directed mutagenesis reactions to generate a library of pathogenic CF variants that were found in the CFTR2 Database(https://www.cftr2.org/). Individual clones from each reaction were generated using the ZymoPURE Plasmid Miniprep Kit (Zymo Research, Irvine, CA) and deep sequenced by shotgun Illumina sequencing to confirm the sequence of each mutated open reading frame and to determine its corresponding 10 base UMI. Plasmids encoding individual variants were pooled with 129 CF variant-encoding plasmids previously described by McKee et al.5 The plasmid pool was then electroporated into electro-competent NEB10b cells (New England Biolabs, Ipswitch, MA), which were then grown in liquid culture overnight and purified using the ZymoPure endotoxin-free midiprep kit (Zymo Research, Irvine, CA). Based on subsequent deep sequencing, the resulting plasmid pool contains 234 variants- the barcode sequence associated with L1065P could not be detected in the final pool. The Bxb1 recombinase expression vector (pCAG-NLS-HA Bxb1) was kindly provided by Douglas Fowler. CFTR variant DNA constructs were stably recombined in modified HEK293T cells containing a genomic Tet-Bxb1-BFP landing pad by co-transfecting with Fugene 6 (Promega, Madison, WI) and Bxb1 recombinase expression vectors. The cells were then incubated at 33 C for 3 days and induced with Doxycycline (2 g/mL) one day after transfection. The cells were then incubated at 37 C for 24 hours prior to the isolation of GFP positive/ BFP negative cells that had undergone recombination using a BD FACS Aria II (BD Biosciences, Franklin Lakes, NJ). These cells were grown in 10 cm dishes with complete media supplemented with doxycycline (2 g/mL) until confluency when they were divided into 15 cm dishes for the three drug trial replicates. Cells were dosed with 3 M VX-661 and/or VX-445 16 hours prior to harvesting when applicable. 7-12 days post-transfection, cells were washed with 1X phosphate buffered saline (AthenaES, Baltimore, MD) and harvested with 1X 0.25% Trypsin-EDTA (Gibco, Grand Island, NY).  CFTR expressed at the plasma membrane of recombinant cells was labeled with a DyLight 550conjugated anti-HA antibody (ThermoFisher, Waltham, MA). Labelled cells were then fractionated into quartiles according to surface immunostaining intensity using a FACS Aria IIu fluorescence activated cell sorter (BD Biosciences, Franklin Lakes, NJ). At least 1 million cells from each fraction were isolated to ensure exhaustive sampling. Fractionated sub-populations were expanded in 10 cm culture dishes prior to harvesting and freezing 15-25 million cells per quartile fraction for the downstream genetic analysis.