Supplementary Figure 1: CRISPR–Cas9-mediated engineering of a neomycin-resistance gene (NeoR) into the Y-chromosome AZFa locus.
a) Step-by-step outline of genome editing strategies used to generate DLD-1 cells and their corresponding clonal derivatives used in this study. NeoR, neomycin-resistance gene. b) Schematic of CRISPR–Cas9-mediated integration of NeoR into the Y-chromosome AZFa locus. The locations of PCR primers used to identify clones with correct NeoR integration are shown. Forward primers outside the left homology arm (HA) produced a PCR product specific for correct integration. As a control, another set of forward primers within the left HA amplified both correct and non-specific integration events. Coordinates represent reference assembly GRCh38. c) PCR using the primers shown in b or the indicated sequence-tagged site (STS). Four correctly targeted clones were identified, of which the indicated clone was chosen for further modifications, as shown in a. d) Representative colony formation plate scans (n = 3 biological replicates) from 3 independent DLD-1 clones carrying a CENP-AC–H3 rescue gene, which were generated as described in a, following the indicated treatment conditions.
