Extended Data Fig. 3: CFTR assembly via BASIS.

a, Recipient cells containing a BASIS assembly BAC with the first section of the CFTR gene and a plasmid encoding Cas9 and lambda red components were mixed with donor cells. Donor cells contained a BASIS BAC encoding the second section of the CFTR gene and a non-transferable F’ plasmid. The donor BAC was conjugated to the recipient cell (A) and recipient cells selected for on tetracycline (+5, tet^R (confers resistance to tetracycline)). Upon induction of protein expression from the helper plasmid, linear dsDNA was excised from the donor BAC (B). The excised DNA inserts into the assembly BAC between HR1 and uHR. Selection on hygromycin (selection for gain of +3) – to select for gain of the selection cassette from the donor BAC, sucrose (selection for loss of −2) – to select for loss of the selection cassette from the assembly BAC and loss of donor BAC backbone, tetracycline (selection for maintenance of +5) – to select for maintenance of the helper plasmid, ensured that only cells with the correctly assembled BAC survive. In step 2, recipient cells containing a BAC with the first and second section of the CFTR gene and a plasmid encoding Cas9, and lambda red components were mixed with donor cells. Donor cells contained a BASIS BAC encoding the third section of the CFTR gene and the non-transferable F’ plasmid. The donor BAC was conjugated to the recipient cell (A) and recipient cells selected for on tetracycline (+5, tet^R (confers resistance to tetracycline)). Upon induction of protein expression from the helper plasmid, linear dsDNA was excised from the donor BAC (B). The excised DNA inserts into the assembly BAC between HR2 and uHR. Selection on chloramphenicol (selection for gain of +2) – to select for gain of the selection cassette from the donor BAC), 4-CP (loss of −3) – to select for loss of the selection cassette from the assembly BAC, streptomycin (loss of −1) – to select for loss of the donor BAC backbone, and tetracycline (maintenance of +5) – to select for maintenance of the helper plasmid, ensured that only cells with the correctly assembled BAC survive. b, Clones from BASIS experiments were picked from the selection plate. They were grown up individually in a 96-well plate and phenotyped for the functionality of selection markers on agar plates. Subsequently, clones that showed the correct growth phenotype and in some cases genotype for the assembly junctions by PCR (step 1: growth on hygromycin, growth on sucrose; no growth on 4-CP, no growth on chloramphenicol, and genotyping for insertion of the second section of CFTR (for primers see Supplementary Data 2); step 2: growth on 4-CP, growth on chlopramphenicol; no growth on hygromycin, no growth on sucrose) were sequenced by NGS. The selectable markers are +3, purple, hyg^R (selected for with hygromycin); −3, orange pheS* (selected against with 4-chlorophenylalanine); +2, green, cat (selected for with chloramphenicol); −2, pink, sacB (selected against with sucrose); −1, yellow rpsL (selected against with streptomycin); +5, dark blue, tet^R (selected for with tetracycline).