Endogenous TINF2-DC mutation leads to the short telomere patient phenotype in hESCs. (A) Experimental overview: generation of isogenic hESC lines. (B) Schematic of TINF2 locus editing by inducing CAS9-mediated double-strand break and homologous recombination in the presence of a repair template including the desired mutation. Successful targeting results in the ablation of the PAM site and de novo generation of a StyI site. (C) Sanger sequencing chromatogram of PCR amplicons of genomic DNA from the WT, heterozygous (het WT/T284R), and homozygous (hom T284R/T284R) hESC lines. (D) IF staining of telomere-binding proteins TRF1 (red) and TIN2 (green) in the WT, het, and hom hESCs. DNA was stained with 4,6-diamino-2-phenylindole (DAPI; blue). Scale bar, 10 μm. (E) Telomere length analysis of WT, het, and hom hESCs at indicated time points after targeting.