C57bl 6 b6 mice

C57BL/6 (B6) mice (7 to 8 weeks old) were purchased from Koatech (Pyeongtaek, Korea). Crif1^fl/fl mice were a generous gift from M. Shong (Chungnam University, Korea) and were crossed with Foxp3^YFP-Cre (Jackson strain no. 016959) or Foxp3^{EGFP-cre-ERT2} (Jackson strain no. 016961) mice. Other purchased mice were B6 CD45.1 congenic (Jackson strain no. 002014), T-bet^fl/fl (Jackson strain no. 022741), Ifng KO (Jackson strain no. 002287), Il4 KO (Jackson strain no. 002253), Rag1 KO (Jackson strain no. 002216), and ROSA26^tdtomato mice (Jackson strain no. 007909) mice. All of these Jackson strain mice were purchased from Jackson Laboratory. Gata3^fl/fl mice were a gift from Professor G. Ryol Lee (Sogang University, Korea). All strains were maintained on a B6 background and crossed as necessary to generate the desired genotypes. Age- and gender-matched male and female mice between 4 and 8 weeks of age were used in the study. Littermates were randomly assigned to experimental groups. All mice were bred and maintained under specific pathogen–free conditions at the Clinical Research Institute, SNUH. All in vivo experiments were approved by the Institutional Animal Care and Use Committee of the Clinical Research Institute, SNUH, and were conducted in accordance with the relevant guidelines and regulations.

C57BL/6 (B6) mice were purchased from Koatech (PyeongTaek, Gyeonggi, Korea). B6.FVIII intron 22 inversion (F8^I22I) and B6.FIX knockout (F9^Mut) were generated by CRISPR-Cas9–based gene editing (fig. S7) (56 (link)). Seven- to 9-week-old male mice were subjected to further experiments. B6 and each hemophilia mice were randomly divided, and half of them were injected LNP packed SpCas9 mRNA and highly modified sgRNA (LNP-CRISPR-mAT) three times with 2-week interval. Injection solution was prepared by mixing 1.2 mpk of LNP-CRISPR-mAT and warm saline up to 600 μl and was injected via the intravenous route. The 600 μl of injection solution could not induce hydrodynamic gene delivery in the liver (fig. S8). B6 mice have been subjected to repeated blood sampling via tail vein for 18 weeks. Hemophilia mice were euthanized at 8 weeks after the first LNP-CRISPR-mAT injection. A total of 450 μl of fresh blood was collected from the inferior vena cava and mixed with 50 μl of 3.2% sodium citrate, and plasma was prepared by collecting supernatant after centrifugation. After blood sampling, each organ was collected without perfusion, a part of the tissues was fixed by formalin, and the remains were used for genomic DNA extraction. This study was approved by the Institutional Animal Care and Use Committees of Seoul National University (SNU-200715-2) and was conducted under approved guidelines.