Base editing induces single-nucleotide changes in the DNA of living cells using a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair1. This genome editing approach has the advantage that it does not require formation of double-stranded DNA breaks or provision of a donor DNA template. Here we report the development of five C to T (or G to A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing 2.5-fold. Additionally, we engineered base editors containing mutated cytidine deaminase domains that narrow the width of the editing window from ∼5 nucleotides to as little as 1–2 nucleotides. We thereby enabled discrimination of neighboring C nucleotides, which would otherwise be edited with similar efficiency, and doubled the number of disease-associated target Cs able to be corrected preferentially over nearby non-target Cs.
Sequence Read Archive
This work was supported by US National Institutes of Health (NIH) R01 EB022376 (formerly R01 GM065400), NIH R35GM118062, F-Prime Biomedical Research Initiative (A28161), and the Howard Hughes Medical Institute. A.C.K. is a Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellow (F32 GM 112366-2). Y.B.K. held a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship (NSERC PGS-D). M.S.P. is an NSF Graduate Research Fellow and was supported by the Harvard Biophysics NIH training grant T32 GM008313.
Supplementary Figures 1–10, Supplementary Notes 1–2, Supplementary Sequences 1–3