Bach1-KO hESCs were generated with CRISPR-Cas9 genome editing technology (40). Single guide RNAs (sgRNAs) targeting the genomic regions of interest were designed with the CRISPR Design Tool ( and synthesized by the Shanghai Sunny Biotechnology Company. Annealed sgRNA oligos were cloned into LentiCRISPR V2 (no. 52961; Addgene, Watertown, MA) and transiently transfected into hESCs via electroporation (no. MPK5000; Life Technologies, Carlsbad, CA) and selected with puromycin (no. ant-pr-5b; InvivoGen, San Diego, CA). Positive clones were identified by sequencing the gene of interest and confirmed by analyzing protein levels. Methods to detect off-target were performed as described previously (41). The cloned sgRNA sequences and off-target primer sequences are listed in table S2.1. DoxBach1 hESCs, including DoxBach1-transfected Bach1-KO hESCs and DoxBach1-transfected WT hESCs, were generated with the PiggyBac transposon system (42). Bach1 complementary DNA (cDNA) was cloned into the vector (pPB-TRE-EGFP-PGK-neoL2) and cotransfected into WT or Bach1-KO hESCs with transactivator and transposase-encoding vectors (pCAG-T7-mPB and pPB-CAG-rtTA-Adv-IRES-Puro); then, transfected cells were isolated with G418 (no. ant-gn-1; InvivoGen, San Diego, CA) and puromycin selection, and clones were chosen for use in subsequent experiments. The PiggyBac transposon system plasmids were provided by J. Na (Tsinghua University, China). Primers for cloning human Bach1 cDNA are listed in table S2.2.

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