Repairing Recessive Compound Heterozygous Mutations In Vivo Via Cas9-Mediated Allelic Exchange

Cas9-mediated gene correction by homology-directed repair (HDR) is a promising therapeutic strategy1-4. However, monogenic genetic diseases typically can be caused by multiple, distinct mutations in the disease gene; treating a population of patients would require a different single-guide RNA (sgRNA) and donor template pair to repair each distinct lesion. Unlike animal models, whose mutations are usually homozygous, compound heterozygous mutations prevail in patients. Here, we report a genome-editing strategy that recombines the pre-existing, correct genetic information present in the two heterozygous alleles into one functional allele, without the use of an exogenous DNA repair template. Recombinant adeno-associated viral (rAAV) vector delivery of Cas9 and a single sgRNA induced allelic exchange and rescued the disease phenotype in a mouse model of hereditary tyrosinemia type I (HT1) that carries compound heterozygous Fah gene mutations. We further showed phenotypic correction in heart for a lysosomal disease mouse model, suggesting that our strategy can target post-mitotic tissues where HDR is generally ineffective. Our strategy can be generalized to repair other compound heterozygous mutations, a genotype category that comprises a large population of patients with recessive genetic disorders.