Guide rna

The plasmid encoding Cas9-2A-GFP was acquired from addgene (#44719; (Ding et al., 2013 (link)). Guide RNA targeting the C-terminus of CDH1 (GGCGGCGAGGACGACTA) was synthesized by Integrated DNA Technologies, cloned into the pGL3-U6-sgRNA-PGK-puromycin vector (addgene #51133; Ferreri et al., 2010 (link)) and sequenced using the RV3 universal primer. To construct the HDR template, homology arms flanking the CDH1 stop codon were independently amplified from genomic DNA and then fused to mRuby2 (addgene #40260; Ferreri et al., 2010 (link)) via overlap extension PCR using the high-fidelity taq polymerase iProof (Bio-Rad). The resulting PCR product was then cloned into the pCR-Blunt II-TOPO cloning vector (Invitrogen) and confirmed by Sanger sequencing. HIESCs were transfected with 2μg of each plasmid using the Amaxa P3 Primary Cell 4D-Nucleofector Kit (Lonza) following the manufacturer’s instructions. Twenty-four hours after transfection, cells were treated with 1mg/ml puromycin for 8 hours to select for positively transfected cells. Cells were expanded for 7 days, then Ruby positive cells were isolated and collected by FACS. Sorted Ruby positive cells were plated at limiting dilution on MEFs in hESC media containing bFGF and surviving colonies were manually separated, clonally expanded, screened for CDH1-Ruby positive fluorescence and karyotyped.

CRISPR lines were generated via Cas9 editing using short single-stranded oligo donors (ssdonors) as previously reported (Ghanta and Mello, 2020 (link)). In brief, two ssdonors were used to introduce synonymous mutations and deletion at target site of 21UR-4864 of w03h9.2. 20 μl injection mix contained pre-assembled Cas9 ribonucleoprotein complex (5 μg Cas9, 2 μg guide RNA, 1 μg tracrRNA) and 1.1 μg ssdonors (Integrated DNA Technologies). The vector pRF4 was used as a co-injection marker (Mello et al., 1991 (link)). Sequences of guide RNA and ssdonors used in this study are listed in Table S5.