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Continuous evolution of base editors with expanded target compatibility and improved activity

Nature Biotechnology volume 37, pages 1070–1079 (2019)Cite this article

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Abstract

Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE–PACE) to improve their editing efficiency and target sequence compatibility. We used BE–PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.

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Fig. 1: Overview of base editing and PACE.
Fig. 2: Design and validation of BE–PACE.
Fig. 3: Design and validation of the split intein BE–PACE selection.
Fig. 4: BE–PACE of APOBEC1, FERNY and CDA1, and characterization of evolved deaminase CBEs in mammalian cells.
Fig. 5: Base editing performance of evolved deaminase CBEs, all codon-optimized in the BE4max architecture, in mammalian cells.
Fig. 6: Performance of evolved CBEs on disease-relevant target sites.

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Data availability

Key plasmids from this work will be available from Addgene (depositor, D.R.L.) and other plasmids are available upon request. All unmodified reads for sequencing-based data in the manuscript are available from the NCBI Sequence Read Archive, accession number PRJNA511456. Figures 4b, 5 and 6, Supplementary Table 3 and Supplementary Figs. 8–14, 16 and 18–22 are based on processing of sequencing data. Protein sequences used for Supplementary Fig. 17 are supplied as Supplementary Data 1.

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Acknowledgements

We thank B. Fu and C. Canavan for assistance with plasmid construction and assays; H. Rees, T. Wang, J. Bessen, A. Badran and P. Lichtor for helpful discussion; K. Clement for CRISPResso2 support; and A. Hamidi for help editing the manuscript. This work was supported by US NIH grant nos. U01 AI142756, RM1 HG009490, R01 EB022376 and R35 GM118062, St. Jude Collaborative Research Consortium, DARPA HR0011-17-2-0049, the Ono Pharma Foundation and HHMI. L.W.K. is an NSF Graduate Research Fellow and was supported by NIH Training Grant no. T32 GM095450. O.S.O. and J.R.H. were supported by NIH DC013521. C.Z. was supported by the Harvard College Research Program. C.W. is the Marion Abbe Fellow of the Damon Runyon Cancer Research Foundation (DRG-2343-18).

Author information

Author notes

  1. B W. Thuronyi

    Present address: Department of Chemistry, Williams College, Williamstown, MA, USA

  2. These authors contributed equally: Luke W. Koblan, Jonathan M. Levy.

Authors and Affiliations

  1. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA

    B W. Thuronyi, Luke W. Koblan, Jonathan M. Levy, Wei-Hsi Yeh, Gregory A. Newby, Christopher Wilson & David R. Liu

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    B W. Thuronyi, Luke W. Koblan, Jonathan M. Levy, Wei-Hsi Yeh, Christine Zheng, Gregory A. Newby, Christopher Wilson & David R. Liu

  3. Program in Speech and Hearing Bioscience and Technology, Harvard Medical School, Boston, MA, USA

    Wei-Hsi Yeh

  4. Department of Neurology, F.M. Kirby Neurobiology Center, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

    Mantu Bhaumik

  5. Departments of Otolaryngology and Neurology, F.M. Kirby Neurobiology Center, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

    Olga Shubina-Oleinik & Jeffrey R. Holt

  6. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    David R. Liu

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Contributions

B.W.T. designed the research, designed and constructed plasmids, and performed PACE and bacterial experiments. L.W.K. and J.M.L. designed and performed HEK cell experiments and analyzed data. J.M.L. designed and performed APOE editing experiments. W-H.Y. designed and performed baringo editing experiments. C.Z. constructed plasmids and performed bacterial experiments for selection development. G.A.N. designed and constructed plasmids for the HEK cell experiment for WFS1 editing. C.W. designed and performed ancestral sequence reconstruction. M.B., O.S-O. and J.R.H. contributed baringo mouse cells. D.R.L. designed and supervised the research. B.W.T., L.W.K. and D.R.L. wrote the manuscript. All authors contributed to editing the manuscript.

Corresponding author

Correspondence to David R. Liu.

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Competing interests

D.R.L. is a consultant and co-founder of Beam Therapeutics, Editas Medicine and Pairwise Plants, companies that use genome editing. D.R.L., B.W.T. and C.W. have filed patent applications on aspects of this work.

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Supplementary information

Complete Integrated SI

Supplementary Discussion 1–6, Supplementary Tables 1–2 and 4–6 and Supplementary Figs. 1–22

Reporting Summary

Supplementary Table 3

Compiled editing data/CRISPResso output tabulated for mammalian cell editing at C bases with measurable edits from any construct

Supplementary Data 1

Structure-guided alignment of APOBEC sequences used in Supplementary Fig. 17 in FASTA format

Supplementary Data 2

Homology model of APOBEC1

Supplementary Data 3

Homology model of FERNY

Supplementary Data 4

Homology model of CDA1

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Thuronyi, B.W., Koblan, L.W., Levy, J.M. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat Biotechnol 37, 1070–1079 (2019). https://doi.org/10.1038/s41587-019-0193-0

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