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

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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Figure 1: SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, and VRER-BE3 mediate efficient base editing at target sites containing non-NGG PAMs in human cells.
Figure 2: Base editors with mutant cytidine deaminase domains exhibit narrowed editing windows.
Figure 3: Genetic variants in ClinVar that can be corrected in principle by the base editors developed in this work.

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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.

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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.

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Correspondence to David R Liu.

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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.

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

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Kim, Y., Komor, A., Levy, J. et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol 35, 371–376 (2017). https://doi.org/10.1038/nbt.3803

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