Pcr blunt 2 topo cloning vector

The plasmid encoding Cas9-2A-GFP was acquired from addgene (#44719, doi: 10.1016/j.stem.2013.03.006). Guide RNA targeting the N-terminus of PROX1 or HES1 was synthesized by Integrated DNA Technologies, cloned into the pGL3-U6-sgRNA-PGK-puromycin vector (addgene #51133, doi: 10.1038/nmeth.2857) and sequenced using the RV3 universal primer. To construct the HDR template, homology arms flanking the PROX1 start codon were independently amplified from genomic DNA and then fused to tdTomato 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.
Human iPSCs were transfected with 2μg of each plasmid using the Lipofectamine 3000 following the manufacturer’s instructions. Twenty-four hours after transfection, cells were sorted by GFP expression to select for positively transfected cells. Clonal cells were expanded for 2 weeks and screened for inserted PROX1-tdTomato or depleted HES1 exon1 sequence and karyotyped.

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.