Fig 2: Genome-editing efficiencies for B2M_e1_1, TGFβR2_e1_1, TGFβR2_e3_1, and TGFβR2_e3_2 TALENs in human iPSC cells, and genotypic characterization of IL-15+/+/TGFβR2−/− iPSC single clones(A) Genomic DNA was extracted from the iPSCs electroporated with B2M_e1_1 TALEN. Then, the B2M exon1 locus was amplified by PCR. Amplicons were sent for Amplicon-EZ sequencing and analyzed to detect the indels. Indel distribution within individual nucleotides was displayed. The left and right TALEN-binding sites are indicated.(B) Genomic DNA was extracted from the iPSC cells electroporated with TGFβR2_e1_1 TALEN. Then, the TGFβR2 exon1 locus was amplified by PCR. Indel distribution within individual nucleotides was displayed. The left and right TALEN binding sites are indicated.(C) Genomic DNA was extracted from the iPSC cells electroporated with TGFβR2_e3_1 TALEN. Then, the TGFβR2 exon3 locus was amplified by PCR. Indel distribution within individual nucleotides was displayed. The left and right TALEN binding sites are indicated.(D) Genomic DNA was extracted from the iPSC cells electroporated with TGFβR2_e3_2 TALEN. Then, the TGFβR2 exon3 locus was amplified by PCR. Indel distribution within individual nucleotides was displayed. The left and right TALEN binding sites are indicated.(E) Mutant percentage and frameshift mutant percentage for B2M_e1_1, TGFβR2_e1, TGFβR2_e3_1, and TGFβR2_e3_2 TALEN in human iPSCs.(F) Genomic DNAs of 32 candidate IL-15 KI iPSC single clones were extracted and were screened for KI by PCR. Top: the locations of the PCR primers for amplifying the KI cassette, IL-15-P2A, at the B2M locus; bottom: PCR products of B2M site-specific IL-15 KIs (Bi-KI, biallelic KI; Mo-KI, monoallelic KI) were displayed together with the positive control (+) and negative control (−) in the gel.(G) A pie chart illustrating the proportions of monoallelic KI, biallelic KI, and the WT among 32 candidate iPSC clones.(H) The genomic DNA of 16 candidate IL-15+/+/TGFβR2−/− iPSC single clones was extracted and screened for TGFβR2 KO by PCR. Top: the locations of the PCR primers for amplifying the editing region at the TGFβR2 locus; bottom: the amplified bands were displayed in the gel. The PCR bands were purified and further characterized by Sanger sequencing.(I) A pie chart illustrating the proportions of homozygous KO, heterozygous KO, and the WT among 12 selected iPSC clones.