Fig. 1.

CRISPR/Cas9-based targeting of human embryonic kidney (HEK-293) cells. A: representative immunofluorescent micrograph shows HEK-293 cells with primary cilia [acetylated (Ac) tubulin: red]; nuclei are stained with DAPI (blue). B: CRISPR/Cas9 targeting of truncating mutations in polycystic kidney and hepatic disease 1 (PKHD1) and polycystic kidney disease 2 (PKD2) genes of the HEK-293 cells. After transfection with CRISPR/Cas9 plasmids (see materials and methods), clones were sorted by green fluorescent protein (GFP) to obtain single-cell clones. Once confluent, these cells were used for genomic DNA isolation and PCR amplification of PKHD1 or PKD2 target sequences to define clones with the sequence modifications at target PKHD1 and PKD2 sites. FACS, fluorescein-activated cell sorting. C: color of culture medium pH indicator (phenol red) changes more rapidly in clones of HEK-293 cells that carry homozygous PKHD1 truncating mutations (yellow medium) than in wild-type (WT) cells (red-pink medium). A similar, although less prominent, change was observed in clones with PKD2 truncating mutations.