Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity

Abstract

Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), which resulted in ABE8e. ABE8e contains eight additional mutations that increase activity (kapp) 590-fold compared with that of ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disruption of regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by the introduction of an additional mutation in the TadA-8e domain. Finally, we show that ABE8e can efficiently install natural mutations that upregulate fetal hemoglobin expression in the BCL11A enhancer or in the the HBG promoter in human cells, targets that were poorly edited with ABE7.10. ABE8e augments the effectiveness and applicability of adenine base editing.

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Fig. 1: Phage-assisted evolution of a deoxyadenosine deaminase.
Fig. 2: Mutations and kinetics of TadA-8e, and editing characteristics of ABE8e in human cells.
Fig. 3: Off-target analysis of ABE8e.
Fig. 4: Adenine base editing with ABE8e at disease-relevant loci in human cells.

Data availability

HTS data have been deposited in the NCBI Sequence Read Archive database (PRJNA589228). All plasmids encoding ABE8e variants in this study will be available through Addgene. A subset of selection plasmids used in this study will be available through Addgene. Other materials are available upon reasonable request.

Code availability

Custom script used to analyze processivity is available in Supplementary Note 1.

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Acknowledgements

This work was supported by US National Institutes of Health awards U01 AI142756, RM1 HG009490, R01 EB022376 and R35 GM118062, St. Jude Collaborative Research Consortium, the Bill and Melinda Gates Foundation and the Howard Hughes Medical Institute (HHMI). M.F.R. was supported by an HHMI Hanna Gray Fellowship. K.T.Z. was supported by Harvard Chemical Biology Training Grant (T32 GM095450). G.A.N was supported by the Helen Hay Whitney Fellowship. C.W. was supported as a Marlon Abbe Fellow of the Damon Runyon Cancer Research Foundation (DRG-2343-18). L.W.K. was supported by a National Science Foundation Graduate Research Fellowship Program. D.E.B. was supported by the National Heart, Lung, and Blood Institute (P01HL053749), Burroughs Wellcome Fund and the St. Jude Children’s Research Hospital Collaborative Research Consortium. We thank S. Miller and T. Wang for providing sgRNA plasmids. We thank A. Raguram for help with computational analyses. We thank J. Doman and A. Raguram for plasmids used during the orthogonal R-loop assay.

Author information

M.F.R. and K.T.Z. conducted the experiments, performed analyses, and wrote the manuscript. E.E., G.A.N, A.L., B.W.T, C.W. and L.W.K. conducted the experiments and performed analyses. J.Z. and D.E.B. provided information on disease loci. J.A.D. provided feedback on biochemical analyses. D.R.L supervised the research and wrote the manuscript. All authors edited the manuscript.

Correspondence to David R. Liu.

Ethics declarations

Competing interests

The authors declare competing financial interests. D.R.L. is a consultant and co-founder of Editas Medicine, Pairwise Plants, Beam Therapeutics, and Prime Medicine, companies that use genome editing. The authors have filed patent applications on evolved ABEs. The Regents of the University of California have patents issued and pending for CRISPR technologies on which J.A.D. is an inventor. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, and Mammoth Biosciences, and a scientific advisory board member of Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Synthego, and Inari; she is a director at Johnson & Johnson. The authors declare no competing non-financial interests.

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Extended data

Extended Data Fig. 1 Mutation table of variants from PANCE and PACE.

Data were obtained by sequencing individual plaques. Conserved mutations are bolded. Mutations that are highlighted in the structure in Fig. 2b are highlighted to match the amino acid positions in the structure. Genotypes in red were tested for base editing activity in mammalian cells.

Extended Data Fig. 2 PACE schedule for deoxyadenosine deaminase evolution.

Lagoon L1 contains host cells harboring P1, P2, and P3e. Lagoons L2 and L3 contain host cells harboring P1, P2, and P3g, which form a more stringent selection circuit than the circuit in lagoon L1. For details on plasmids, see Supplementary Table 1. The stringency of the ABE selection was further modulated by increasing the lagoon flow rate (dashed lines). For the first 12 hours, gene III was expressed by the addition of anhydrotetracycline to enable genetic drift in the absence of selection pressure12,13.

Extended Data Fig. 3 Titration data at eight editor doses comparing base editing efficiencies for ABE8e and ABE8e-dimer at three sites in HEK293T cells.

Base editing with ABE8e and ABE8e-dimer in HEK293T cells at three genomic sites in HEK293T cells. Transfections were performed with constant amount of sgRNA plasmid but eight varying doses of ABE plasmid. For all plots, dots represent individual biological replicates and bars represent mean±s.d. of three independent biological replicates.

Extended Data Fig. 4 TadA-8e V106W analysis for SaCas9 and LbCas12a.

a, DNA editing comparing SaABE7.10, SaABE8e, and SaABE8e(TadA-8e V106W) at four genomic sites in HEK293T cells. b, DNA editing comparing LbABE7.10, LbABE8e, and LbABE8e(TadA-8e V106W) at six genomic sites in HEK293T cells. For all plots, dots represent individual biological replicates and bars represent mean±s.d. of three independent biological replicates.

Supplementary information

Supplementary Information

Supplementary Figs. 1–17, Tables 1–5, Note 1 and Sequences 1

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Richter, M.F., Zhao, K.T., Eton, E. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0453-z

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