Letter | Published:

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

Nature Biotechnology volume 35, pages 371376 (2017) | Download Citation

Abstract

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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Acknowledgements

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

Affiliations

  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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Contributions

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.

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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.

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    Supplementary Text and Figures

    Supplementary Figures 1–10, Supplementary Notes 1–2, Supplementary Sequences 1–3

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https://doi.org/10.1038/nbt.3803

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