CRISPR-Cas9 Knockout Protocol for HCT-Oxa-c Cells

The sgRNA-23b and sgRNA-27b were designed using the Benchling program (Supplementary Figure S7) and inserted into pSpCas9(BB)-2A-Puro (Addgene 62988) as described previously [22 (link)]. HCT-Oxa-c cells (15 × 10⁴ cells/well) were seeded in 24-well plates 24 h before transfection and constructs were transfected into HCT-Oxa-c cells using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 48 h of transfection, cells were incubated for 48–72 h with 2 µg/mL puromycin (Sigma). Then puromycin-resistant cells were isolated through serial dilutions, seeding one cell per well of 96-well plates. Cells were grown in an incubator for 1–2 weeks, when cultivated into 24-well plates. Some of the cells were collected into tubes and indels were detected as described previously using polyacrylamide gel electrophoresis [23 (link)]. PCR was carried out with the Phire Tissue Direct PCR master Mix (Thermo Fisher Scientific). The list of primers and single-guide RNA (sgRNA) sequences (Metabion) are provided in Supplementary Table S15.