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Figure 1

From: Generation and characterization of human U-2 OS cell lines with the CRISPR/Cas9-edited protoporphyrinogen oxidase IX gene

Figure 1

Establishing PPO-KO cell lines by CRISPR/Cas9 editing of the PPOX gene. (a) Schematic representation of PPOX gene disruption. Positions of CRISPR/Cas9 target sites are marked by red crosses. Sequences amplified by PCR for detection of heteroduplexes and allele genotyping are highlighted in yellow. (b) Analysis of PPOX allele heteroduplexes from CRISPR/Cas9 edited cell lines. Regions encompassing CRISPR recognition sites were PCR-amplified, annealed, and separated by PAGE with GelRed staining. Representative samples of CRISPR/Cas9 clones of PPOX-Arg38, PPOX-Trp227, and PPOX-Cys459 loci are shown. (c) The PPO enzymatic activity in cell lysates of PPO-KO and control clones was determined by quantifying the conversion of protoporphyrinogen IX to protoporphyrin IX. PPO oxidase activity in individual clones was normalized to the total protein content and is shown as a fraction of the activity of the U-2 OS parental cell line. Cell clones selected for further analysis are highlighted by grey background. Data represent mean (± S.D.); n = 3.

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