Fig. S1.

PB-CRISPR vectors and validation by targeting Tet1 and Tet2 in mouse iPS cells. (A) PB-based CRISPR vectors. pCRISPR-sg4, sgRNA-expressing vector with neo gene; pCRISPR-sg5, sgRNA-expressing vector with puromycin gene. (B) pCRISPR-S10, PB plasmid expressing Doxycycline-inducible Cas9; pCRISPR-sg6-Tet1/Tet2, pCRISPR-sg6-based plasmids expressing Tet1 or Tet2 sgRNA. (C) PCR-RFLP analysis of Tet1/Tet2 loci targeted by pCRISPR-sg6-Tet1/Tet2. Expected mutations would eliminate the SacI or EcoRV site in Tet1 and Tet2, respectively. The target regions (∼500 bp) of Tet1 or Tet2 were amplified by PCR. PCR products were digested with corresponding enzymes. Results showed the successful targeting in Tet1-clone 1, Tet1-clone 2, and Tet2-clone 2. (D) Sequencing results of Tet1/Tet2 sgRNA targeted loci. Sequencing results for Tet1-clone 1 revealed a 4-bp deletion in one allele and a 1-bp deletion in another, resulting in elimination of the SacI site. Sequencing results for Tet1-clone 2 revealed mutations in both alleles, with a 3-bp deletion in one and a 1-bp insertion in another, resulting in elimination of the SacI site. Sequencing results for Tet2-clone 2, with an 8-bp deletion in one allele and a 14-bp deletion in another, resulting in elimination of the EcoRV site.