Generation of intron 22 inversion based hemophilia A humanized mice and gene correction with Adeno Associated Virus and CjCas9

Abstract

Abstract

Generation of intron 22 inversion-based hemophilia A humanized mice and gene correction with Adeno Associated Virus and CjCas9

Yoon Hoo Lee Department of International Agricultural Technology The Graduate School Seoul National University

Hemophilia A (HA) is caused by mutated Factor VIII (F8) by nonsense or missense mutation, small indel, splice site mutation, large deletion, or inversion. In particular, intron22 inversion (inv22) derived HA accounts for almost half of patients with severe HA. Despite the need of inversion based mouse model, there have been no suitable inv22 HA mouse model. In this study, I presented genetically induced inversion by using CRISPR/Cas9. In embryo, Non-homologous end joining (NHEJ) mediated inversion was shown at 13.1% efficiency. Homology direct repair (HDR) mediated inversion exhibits 43.8% efficiency, and then, I successfully generated inversion based HA humanized (Hu-inv22 HA) mice at 53.8% efficiency. Despite breakage in flank of inversion junction site, all mutated mouse have inverted F8, and human sequence in inversion junction, which I insert in inversion junction for gene correction. I also observed hemophilia A related symptom, such as no detectable F8 in liver and excessive bleeding. In addition, we revert inverted F8 in Mouse Embryonic Fibroblast (MEF) by using AAV containing CjCas9 targeting human sequence in inversion junction. Therefore, we confirmed that AAV can be applicable to correction of the inversion case. The results suggests that Hu-inv22 HA mouse can be used in diverse study for inversion based HA, and additionally AAV can be suitable vector for correction of HA