Figure 1.

Deletion of ORC5 in HCT116 p53^−/− cells. A, CRISPR/Cas9 biallelic targeting strategy for ORC5. ORC5 sgRNA target first methionine located upstream of Walker A motif. Second methionine locates at 133 amino acid (aa). B, DNA sequences of WT ORC5 exon 1 and three ORC5Δ clones obtained from genomic sequencing and by sequencing of cDNA. First methionine site is mutated in both allele of mutant clones. C, verification of ORC5 antibody. Recombinant ORC5 proteins with Myc tag were expressed and blotted with indicated antibodies. Ponceau S staining shows equal loading of lysate. D, Western blotting of ORC5 in the ORC5Δ clones shows that full-length and truncated ORC5 proteins are undetectable.