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Cited by 68CRISPR and Genetic EngineeringRNA regulation and diseaseEnzyme Catalysis and Immobilization

Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference

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Zhang, Emily, Neugebauer, Monica E., Krasnow, Nicholas A., Liu, David R.·2024·Nature Communications
DOI 10.1038/s41467-024-45969-7PMC PMC10894238
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Abstract

TadA-derived cytosine base editors (TadCBEs) enable programmable C•G-to-T•A editing while retaining the small size, high on-target activity, and low off-target activity of TadA deaminases. Existing TadCBEs, however, exhibit residual A•T-to-G•C editing at certain positions and lower editing efficiencies at some sequence contexts and with non-SpCas9 targeting domains. To address these limitations, we use phage-assisted evolution to evolve CBE6s from a TadA-mediated dual cytosine and adenine base editor, discovering mutations at N46 and Y73 in TadA that prevent A•T-to-G•C editing and improve C•G-to-T•A editing with expanded sequence-context compatibility, respectively. In E. coli , CBE6 variants offer high C•G-to-T•A editing and no detected A•T-to-G•C editing in any sequence context. In human cells, CBE6 variants exhibit broad Cas domain compatibility and retain low off-target editing despite exceeding BE4max and previous TadCBEs in on-target editing efficiency. Finally, we show that the high selectivity of CBE6 variants is well-suited for therapeutically relevant stop codon installation without creating unwanted missense mutations from residual A•T-to-G•C editing.

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Authors
Zhang, E., Neugebauer, M. E., Krasnow, N. A., & Liu, D. R.
Affiliations
Broad Institute (US)
Corresponding author
Liu, David R.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2024-02-24
Volume
15
Issue
1
Pages
1697-1697
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-024-45969-7
PMC
PMC10894238
OpenAlex
W4392129257
Cited by
68
References
53
Citations by year
2026: 26 · 2025: 35 · 2024: 7
FWCI
9.26
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (R35GM118062)
  • U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (U01AI142756)
  • U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (R01EB027793, R01EB031172)
  • U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (R01HL156647)
  • Howard Hughes Medical Institute (Liu investigatorship)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
cytosine base editors, A-to-I RNA editing, off-target effects
MeSH terms
Gene Editing, Bacteriophages, Cytosine, Escherichia coli, Humans, CRISPR-Cas Systems

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