Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction

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Abstract

Base editors enable targeted single-nucleotide conversions in genomic DNA. Here we show that expression levels are a bottleneck in base-editing efficiency. We optimize cytidine (BE4) and adenine (ABE7.10) base editors by modification of nuclear localization signals (NLS) and codon usage, and ancestral reconstruction of the deaminase component. The resulting BE4max, AncBE4max, and ABEmax editors correct pathogenic SNPs with substantially increased efficiency in a variety of mammalian cell types.

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Figure 1: Identifying and addressing factors that limit base-editing efficiency in mammalian cells.
Figure 2: Properties of optimized AncBE4max, BE4max, and ABEmax compared to those of BE4 and ABE7.10.

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Acknowledgements

This work was supported by the Ono Pharma Foundation, DARPA HR0011-17-2-0049, US NIH RM1 HG009490, R01 EB022376, and R35 GM118062, and HHMI. Flow cytometry was supported by NCI P30CCA14051. L.W.K. is an NSF Graduate Research Fellow and was supported by NIH Training Grant T32 GM095450. J.L.D. gratefully acknowledges graduate fellowship support from the NSF and Hertz Foundation. We thank J. Coller, G. Hansen, M. Weiss, and A. Sharma for helpful discussions.

Author information

L.W.K., J.L.D., C.W., J.M.L., T.T., G.A.N., and J.P.M. generated reagents and conducted experiments. C.W. and A.R. performed computational analyses. D.R.L. supervised the research. All authors contributed to writing the manuscript.

Correspondence to David R Liu.

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Competing interests

D.R.L. is a consultant and co-founder of Editas Medicine, Pairwise Plants, and Beam Therapeutics, companies that use genome editing. L.W.K., J.L.D., C.W., and D.R.L. have filed patent applications on aspects on this work. The authors declare no competing non-financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–17, Supplementary Note 1, Supplementary Sequences 1–5 (PDF 2354 kb)

Life Sciences Reporting Summary (PDF 163 kb)

Supplementary Data 1

MAFFT alignment APOBEC homologs in FASTA format. (TXT 112 kb)

Supplementary Data 2

Flow cytometry gating examples for all cell types used (PDF 3674 kb)

Supplementary Data 3

APOBEC tree in Newick format (TXT 32 kb)

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Koblan, L., Doman, J., Wilson, C. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol 36, 843–846 (2018) doi:10.1038/nbt.4172

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