(Supplementary Figs. 1–4). Previous studies suggested that CRISPR-Cas systems may tolerate sequence mismatches distal from the protospacer adjacent motif (PAM) at the 5о end of sgRNAs, which would probably induce off-target mutations. To detect offtarget effects of the sgRNA: Cas9 constructs in vivo, we screened the genome of 13 triplemutated rats for all off-target sites with more than 14-bp sequence identity to the six sgRNAs. Only four such sites were identified for all six sgRNAs (Supplementary Fig. 7). The SURVEYOR assay revealed that only one potential site was mutated by sgTet1-1 in all the examined triple-mutated rats (Supplementary Fig. 8). The high birth rate and survival rate of the mutant rats (except for the neonatal lethal gene Tet3) indicated that the sgRNA: Cas9 construct had very low toxicity to rat embryos (Table 1). Successful germline transmission of genetic mutations is essential for establishing genetically modified animal models. To test the transmission ability of the mutation, we produced fertilized embryos with sperm from Tet1/Tet2 double-mutant rats and wildtype oocytes. Of seven embryos successfully assayed by Sanger sequencing, we identified five inherited mutations in the Tet2 site only, and two inherited mutations in both the Tet1 and Tet2 sites, indicating that mutations in the founder rats were transmitted to the next generation (Supplementary Fig. 9).