Figure 2.

CRISPR gene editing of the HIF1A gene in HUVECs. (a) Graphical illustration of the HIF1A gene, target exons, and gRNAs. The top panel shows the length of the HIF1A gene (chromosome 14 at position 61.695.512–61.748.259). Three isoforms of the gene are known, which contain 14–15 exons (indicated by the grey boxes). The green, red, and yellow, boxes visualize the three target consecutive exons (exon two, −three, and −four). The sequence of HIF1A gRNA 1, −2, and −3 is captured in green, red, and yellow bars. (b) PCR amplification of the CRISPR gene editing of HUVECs. Visualization of the gel electrophoresis of the amplified DNA from HIF1A gRNA 1, −2, and −3 gene edited HUVECs, HIF1A WT (wild type) HUVECs, HEK293T cells, and the lysis buffer. The 100 bp gene marker is shown in the first well. The green frame indicates the samples amplified by the HIF1A F1/R1 primer sets. (c) CRISPR gene editing efficiency of the three different HIF1A gRNAs. Quantification of the gene editing efficiency of the HIF1A gRNA 1, −2, and −3 and the WT. Values are presented as the mean percentage of indels $\pm$ STD. (d) Representative visualization of the Sanger sequencing results for each HIF1A gRNA. For each HIF1A gRNA, the WT HUVECs sequence is shown in the bottom row. The gRNA sequence is underlined with black. The PAM sequence is underlined in dotted red. The editing site is marked by the black dotted vertical line. The sequence of the gene-edited part is shown by wave plots. The nucleotides are color coded as G = guanine, C = cytosine, T = thymine, and A = adenine. (b–d) Experiments on HUVECs from three biological donors at p. 2–4 (n = 3). HIF1A gRNA 1–3 experiments are performed with one biological donor at p. 4. HIF1A gRNA 1 experiments were repeated to achieve three biological replicates.