Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins.

ABSTRACT

Many genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of CG to GC Base Editors to create single-base genomic transversions in human cells. Our CG to GC Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform CG to GC editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). CG to GC Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.