In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA

Base editors are RNA-programmable deaminases enabling precise single-base conversions on genomic DNA. However, off-target activity is a concern in the potential use of base editors to treat genetic diseases. Here, we report unbiased analyses of transcriptome-wide and genome-wide off-target modifications effected by cytidine base editors in the liver of mice with phenylketonuria. The intravenous delivery of intein-split cytosine base editors via dual adeno-associated viruses led to the repair of the disease-causing mutation without generating off-target mutations in the RNA and DNA of the hepatocytes. Moreover, the transient expression of a cytidine base editor mRNA and a relevant single-guide RNA intravenously delivered via lipid nanoparticles led to ~21% on-target editing and to the reversal of the disease phenotype, also without detectable transcriptome-wide and genome-wide off-target edits. Our findings support the feasibility of therapeutic cytidine base editing to treat genetic liver diseases.