The safety of highly stable Cas9 in mice

Twenty-four male C57BL/6 mice (5 weeks old) were randomly divided into three groups (NC, Cas9, and HSCas9 groups). NC group were hydrodynamically injected with the plasmid pBB4.5-1.3HBV, PX458-Cas9, and pU6 plasmids. Cas9 group were hydrodynamically injected with the plasmid pBB4.5-1.3HBV, PX458-Cas9, and pU6-t-g1-t-g2-t plasmids. HSCas9 group were hydrodynamically injected with the plasmid pBB4.5-1.3HBV, PX458-HSCas9, and pU6-t-g1-t-g2-t plasmids. At 7 days post of hydrodynamic injection, mouse livers were collected, and three mice were randomly selected from each group to extract genomic DNA from liver tissue, followed by whole genome sequencing.Above experiments were approved by the Ethics Committee of Peking University and were carried out according to the guidelines established by the Institutional Animal Care and Use Committee at Peking University Health Science Center.