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Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity
Open accessAbstract
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 (k app) 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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“ABE8e has an “editing window,” the nucleotides accessible for deamination, of positions 4–9 in the protospacer (11–16 bases upstream from the PAM).16”
From Adenine base editor for knockout of proteins: A practical guide from design to analysis with updated MultiEditRbatch (2026) · cites this paper as [16]
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“Nevertheless, accumulative studies have proved that sgRNA-independent off-target effects also exist, urging unbiased experimental detection and validation (Richter et al., 2020; O'Geen et al., 2015).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Richter et al., 2020
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“The sgRNA-independent DNA editing is likely deposited on loci where DNA unwinds into an R-loop so single-strand genomic DNA is exposed to deaminases”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Richter et al., 2020
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“ABE8e, a variant published in 2020, exhibits a 590-fold increase in activity due to numerous mutations in the TadA deaminase domain.16”
From Adenine base editor for knockout of proteins: A practical guide from design to analysis with updated MultiEditRbatch (2026) · cites this paper as [16]
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“Truncated gRNA: truncated by 2-3bp at 5’ end | Decreased off-target effects”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [72]
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“Extended gRNA: GGX20 | Decreased off-target effects”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [72]
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Additional file 2 of Quantification of evolved DNA-editing enzymes at scale with DEQSeq
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Additional file 1 of Quantification of evolved DNA-editing enzymes at scale with DEQSeq
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Additional file 3 of Quantification of evolved DNA-editing enzymes at scale with DEQSeq
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Additional file 4 of Quantification of evolved DNA-editing enzymes at scale with DEQSeq
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Additional file 5 of Quantification of evolved DNA-editing enzymes at scale with DEQSeq
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Metadata
- Authors
- Richter, M. F., Zhao, K. T., Eton, E., Lapinaite, A., Newby, G. A., Thuronyi, B. W., Wilson, C., Koblan, L. W., Zeng, J., Bauer, D. E., Doudna, J. A., & Liu, D. R.
- Affiliations
- Broad Institute (US)
- Boston Children's Hospital (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Liu, David R.
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2020-03-16
- Volume
- 38
- Issue
- 7
- Pages
- 883-891
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC7357821
- OpenAlex
- W3012327875
- Cited by
- 1,020
- References
- 60
- Citations by year
- 2026: 158 · 2025: 196 · 2024: 192 · 2023: 192 · 2022: 148 · 2021: 103 · 2020: 29
- FWCI
- 38.37
- Type
- journal article
- Language
- en
- Update notices
- correction (2020-05-20)
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion, cc-by)
- Funders
- U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (P01HL053749)
- Division of Intramural Research, National Institute of Allergy and Infectious Diseases (U01 AI142756)
- U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (RM1 HG009490)
- U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (R01 EB022376)
- U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (R35 GM118062)
- 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 › Genetics › Virus-based gene therapy research
- Keywords
- adenine base editing, ABE8e, phage-assisted continuous evolution, Cas12, processivity, off-target editing, fetal hemoglobin, PAM site
- MeSH terms
- Gene Editing, Adenine, Adenosine Deaminase, Bacteriophages, DNA, Humans, Mutation, RNA, Mutagenesis, HEK293 Cells, CRISPR-Cas Systems
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