Generation of CRISPR knockout cell lines

sgRNAs targeting the following sequences are listed in (Table. 1). Guides were cloned into px459v2 (Addgene #62988), px458 (Addegene #48138), LentiCRISPRv2 (Addgene #52961), Lenti-sgRNA-Hygro (Addgene #104991) or Lenti-sgRNA-Puro (Addgene #104990) as indicated (Table. 1). ALC1^+/+ and ALC1^−/− eHAP were generated by transducing eHAP iCAS9 generated as described above with NT gRNA or ALC1 gRNA cloned into Lenti-sgRNA-Hygro (Table. 1). Cells were selected in Hygromycin at a concentration of 400 μg ml⁻¹. The resulting NT gRNA and ALC1 gRNA iCAS9 cell lines were then banked. To make individual knockout clones, cells were treated with 1 μg/mL Dox for 72 h and then seeded as single cell clones by limiting dilution. The resulting plates were duplicated and screened using IF for ALC1. Knockout clones were then confirmed by immunoblotting against ALC1. ALC1^−/− U2OS Flp-In T-REx cell lines were generated by the transient transfection of cells with px459 containing guides against ALC1. Clones were isolated and screened as above. Inducible CRISPR knockout cell lines were generated by transducing ALC1^+/+ and ALC1^−/− iCAS9 cells with lentivirus produced from the sgRNA constructs listed in (Table. 1) followed by antibiotic selection. Knockout of target proteins was confirmed by immunoblotting following 72 h 1 μg/mL Dox. SMUG1 and APEX1 KO cell lines were created by transiently transfecting ALC1^+/+ and ALC1^−/− iCAS9 cells with APEX1 LentiGuide puro and SMUG1 LentiGuide puro. Cells were pulsed with 0.4ug/mL puro for 2 days and Cas9 expression was induced by treating with 1μg/mL Dox for 72 h. Clones were isolated and screened as above.