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Directed evolution of adenine base editors with increased activity and therapeutic application

Nature Biotechnology volume 38, pages 892–900 (2020)Cite this article

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Abstract

The foundational adenine base editors (for example, ABE7.10) enable programmable A•T to G•C point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG protospacer adjacent motif (PAM) sites, ABE8s result in ~1.5× higher editing at protospacer positions A5–A7 and ~3.2× higher editing at positions A3–A4 and A8–A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2 with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98–99% target modification, which is maintained when multiplexed across three loci. Delivered as messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

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Fig. 1: Eighth generation adenine base editors mediate superior A•T to G•C conversion in human cells.
Fig. 2: Cas9 PAM-variant ABE8s and catalytically dead Cas9 ABE8 variants mediate higher A•T to G•C conversion than corresponding ABE7.10 variants in human cells.
Fig. 3: A•T to G•C conversion and phenotypic outcomes in primary human cells.
Fig. 4: Whole transcriptome and whole genome sequencing data from cells treated with base editor mRNAs.

Data availability

Plasmids encoding the core ABE8s used in this work are available from Addgene. High-throughput sequencing data are deposited in the NCBI Sequence Read Archive (PRJNA574182). Source data are available online for Figs. 1–4 and Supplementary Figs. 3–26 and 28.

Code availability

All software tools used for data analysis are publicly available. Detailed information about versions and parameters used, as well as shell commands, are provided in Supplementary Note 4.

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Acknowledgements

We thank S. Cavnar for support with creating digital renderings of base editing mechanisms. We acknowledge D. Levasseur and B. Yan for technical advice and support with CD34+ experiments. We thank B. Gantzer, J. Decker and M. Humes for NGS support. We recognize and thank S. Haskett for his FACS expertise and sorting mammalian cells used in WGS experiments.

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  1. Jonathan Yen

    Present address: Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN, USA

Authors and Affiliations

  1. Beam Therapeutics, Cambridge, MA, USA

    Nicole M. Gaudelli, Dieter K. Lam, Holly A. Rees, Noris M. Solá-Esteves, Luis A. Barrera, David A. Born, Aaron Edwards, Jason M. Gehrke, Seung-Joo Lee, Alexander J. Liquori, Ryan Murray, Michael S. Packer, Conrad Rinaldi, Ian M. Slaymaker, Jonathan Yen, Lauren E. Young & Giuseppe Ciaramella

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Contributions

N.M.G. conceived and directed the work, conducted evolution and Hek293T experiments, performed analyses, and wrote the manuscript. D.K.L. and N.M.S.-E. conducted mammalian cell transfections. H.A.R. conducted all off-target studies and analyses except for whole transcriptome sequencing and WGS analyses, which were conducted by L.E.Y. M.S.P. and J.Y. designed CD34+ experiments and synthesized mRNA constructs. C.R., J.Y., and A.J.L. carried out all CD34+ experiments and associated UHPLC procedures. A.E., R.M. and J.M.G. conducted all T-cell experiments and analyses. L.A.B. conducted all statistical analyses of NGS data and generated figures. D.A.B. created PyMol figures. S.-J.L. and I.M.S. designed truncation experiments. G.C. supervised the research. L.E.Y. processed, analyzed and generated figures for whole transcriptome sequencing and WGS. N.M.G., H.A.R., M.S.P., J.M.G., L.A.B. and G.C. all edited the manuscript.

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Correspondence to Nicole M. Gaudelli or Giuseppe Ciaramella.

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All authors were employees of Beam Therapeutics when the work was conducted and are shareholders in the company. Beam Therapeutics has filed patent applications on this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–30, Supplementary Tables 1–27, Supplementary Notes 1–6, Supplementary Sequences 1–9 and Supplementary References

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Gaudelli, N.M., Lam, D.K., Rees, H.A. et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat Biotechnol 38, 892–900 (2020). https://doi.org/10.1038/s41587-020-0491-6

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