Crrna

To generate NODAL-knockout (KO) hPSCs, a 58-bp portion of genomic DNA within NODAL exon 1 was deleted by CRISPR/Cas9 using two crRNA purchased from Thermo Fisher Scientific [NODAL_crRNA_1: 5’-AGGCUCAGCAUGUACGCCAG-3’; NODAL_crRNA_2: 5’-AGACAUCAUCCGCAGCCUAC-3’] (Figures S7A–S7C). Deplexes of crRNA:tracrRNA were prepared using a standard protocol and introduced into H9 hPSCs with the Cas9 enzyme and the pCXLE-EGFP expression plasmid (a gift from Shinya Yamanaka; Addgene plasmid # 27082; RRID: Addgene_27082) for constitutional expression of EGFP using the NEON electroporation system (Thermo Fisher Scientific). EGFP-expressing single cells were collected and seeded onto Matrigel-coated 96-well plates by fluorescence-activated cell sorting (FACSAria Fusion, BD Biosciences) with CloneR single-cell culture supplement diluted with mTeSR Plus medium (STEMCELL Technologies). To detect the anticipated deletion, genomic DNA was isolated from single-cell derived clones and subjected to PCR using the following primers designed for amplification of NODAL exon 1 [Forward Primer: 5’-CTTCCTTCTGCACGCCTGGTGG-3’; Reverse Primer: 5’-CCAACCCACAGCACTTCCCGAG-3’]. Resulting amplicons were subjected to Sanger sequencing using a primer 5’-CTTCCTTCTGCACGCCTGGTGG-3’. ESI-017 NODAL-KO hPSC line is a generous gift from Aryeh Warmflash at Rice University (Chhabra et al., 2019 (link)).