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 C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C 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 C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C 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.
Highlights
Many genetic diseases are caused by single-nucleotide polymorphisms
We demonstrate that this class of C:G to G:C Base Editors (CGBEs) edits C:G to G:C (Fig. 1a), which potentially opens up treatment avenues to 11% to 40% (CGBE with cytidine base editors (CBEs)/adenine base editors (ABEs)) of the disease-associated SNPs in ClinVar (Supplementary Table 1)
Since no effective means of C:G to G:C base editing was known at the time, and BE3 has been observed to effect this reaction as a byproduct of C:G to T:A editing[4], we used BE3 as a benchmark for our CGBEs
Summary
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. Two recent reports capitalized on this observation and demonstrated that fusion of APOBEC-nCas[9] to uracil DNA glycosylase (UNG) – which induces abasic sites – results in C:G to G:C editing in mammalian cells[6,7]. We demonstrate a distinct CGBE architecture that manipulates the BER pathway downstream of abasic site creation. We demonstrate that this class of C:G to G:C Base Editors (CGBEs) edits C:G to G:C (Fig. 1a), which potentially opens up treatment avenues to 11% (singular CGBE) to 40% (CGBE with CBE/ABE) of the disease-associated SNPs in ClinVar (Supplementary Table 1)
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