Fig 2: Off-targets identified from Slc17a5 F0 mice. Sequencing traces of PCR products encompassing the target regions of Virma a, Ctla-4 b, or Slc17a5 c in the tissues or Treg cells of representative mutant mice produced by the OSTCM method. Overlapping sequencing traces indicate the presence of more than one allele among the mutant mice. Editing (indel) efficiencies assessed by TIDE analysis in each tissue is shown to the right of each trace. d Venn diagram displaying the overlap of all single-nucleotide variants detected in whole-genome sequencing data from three mutant mice (#10, #17, and #18). e The top ten potential off-target sites of CRISPR-Cas9 for sgRNA. Red nucleotides represent a mismatch compared with the on-target sequences. Ten selected potential off-target sites analyzed with Sanger sequencing of the livers of the three mutant mice from d

Fig 3: Heritable F0 mice with Slc17a5 or Ctla-4 insertion/deletion mutations. a Schematic diagram of the sgRNA targeting site in the mouse Slc17a5 locus. PAM = protospacer adjacent motif. b PCR sequences encompassing the Slc17a5 target region from wild-type (wt) mice and from representative mutant mice (#4 and #16) generated by two methods of microinjection: (1) into zygotes using sgRNA; and (2) into one blastomere of two-cell stage embryos using sgRNA. PCR products were sequenced and those showing overlapping sequencing traces were cloned. The subsequent individual clones were then sequenced. Mutated bases are labeled in red and deleted nucleotides are indicated by hyphens. Numbers and percentages of the mutated bases are listed on the right. c Sequencing traces of PCR products from representative mutant mice generated by the aforementioned two microinjection methods. Overlapping sequencing traces among the mutant mice indicate the presence of more than one allele among these mice, compared with wt mice. The mutations’ start positions are indicated by black arrows. d Germline transmission of the Slc17a5 lethal mutations in F1 progeny is shown by sequencing traces of PCR products encompassing the Slc17a5 target region from a representative mutant F1 mouse. Overlapping sequencing traces indicate heterozygous mice. The black arrow shows the mutation starting position. e Schematic diagram of the sgRNA targeting site in the mouse Ctla-4 locus. f Sequencing traces of PCR products encompassing the Ctla-4 target region from wt mice and from a representative mutant F0 mouse. The mutant mouse’s overlapping sequencing traces indicate the presence of more than one allele, compared with wt mice. The mutation start position is indicated by a black arrow. g PCR sequences of the Ctla-4 target region of wt mice and a representative mutant mouse generated by microinjection into one blastomere of two-cell embryos. The mutated base is labeled in red and deleted nucleotides are indicated by hyphens