Gene Editing in Hemophilic Mice

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.