Deep sequencing reads for cytidine base editing in HT1 mouse model

In this study, we generated a novel hereditary tyrosinemia type 1 (HT1) mouse model which contains a start codon mutation in the fumarylacetoacetate hydrolase (Fah) gene by using an adenine base editor (ABE). To investigate the feasibility of base editing for recombinant adeno-associated virus (rAAV) mediated gene therapy, an intein-split cytidine base editor (BE4) was developed. BE4 efficiently induced C>T conversion to restore the start codon and ameliorated HT1 in mice, but an undesired bystander mutation abolished the effect of on-target editing. To solve this problem, an upstream sequence was targeted to generate a de novo in-frame start codon to initiate the translation of Fah. After treatment, almost all C>T conversions created a start codon and restored Fah expression and translation which efficiently ameliorated the disease without inducing off-target mutations. Our study demonstrated that base editing-mediated creation of de novo functional elements would be an applicable new strategy for genetic disease therapy.