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Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions

Nature Biotechnologyvolume 35pages371376 (2017) | Download Citation


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.

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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.

Author information


  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    • Y Bill Kim
    • , Alexis C Komor
    • , Jonathan M Levy
    • , Michael S Packer
    • , Kevin T Zhao
    •  & David R Liu
  2. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    • Y Bill Kim
    • , Alexis C Komor
    • , Jonathan M Levy
    • , Michael S Packer
    • , Kevin T Zhao
    •  & David R Liu
  3. Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA

    • David R Liu


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Y.B.K., A.C.K., J.M.L., and K.T.Z. conducted the experiments. M.S.P. performed computational analyses. D.R.L. supervised the research. All authors contributed to writing the manuscript.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Competing interests

D.R.L. is a consultant and co-founder of Editas Medicine, a company that seeks to develop genome-editing therapeutics. Y.B.K., A.C.K., and D.R.L. have filed patent applications on base editing.

Corresponding author

Correspondence to David R Liu.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–10, Supplementary Notes 1–2, Supplementary Sequences 1–3 (PDF 661 kb)

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