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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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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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).
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.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Discussion 1–6, Supplementary Tables 1–2 and 4–6 and Supplementary Figs. 1–22
Compiled editing data/CRISPResso output tabulated for mammalian cell editing at C bases with measurable edits from any construct
Structure-guided alignment of APOBEC sequences used in Supplementary Fig. 17 in FASTA format
Homology model of APOBEC1
Homology model of FERNY
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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