Continuous evolution of base editors with expanded target compatibility and improved activity
Open accessAbstract
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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Evolved base editor variants such as EvoFERNY and CBE6-variants no longer have the GC sequence context restriction.
“The evolved variants such as EvoFERNY and CBE6-variants do no longer have this sequence context.”
From Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools (2024) · cites this paper as [25]
The Discussion (page not available) says evoFERNY-BE4max and evoAPOBEC1-BE4max showed no apparent bias against GC targets at the sites tested. The paper does not identify a family called “CBE6-variants,” and its finding is limited to tested sites rather than establishing that the restriction is absent in every context. (LLM, conf=0.84)
Experimental — accuracy not yet measuredEditors such as CBE6b or EvoFERNY do not have the sequence context limitation of not editing when a G precedes the target C.
“For this reason, make sure to always be updated with the literature and find editors that do not have such a sequence context, such as CBE6b or EvoFERNY.25,53”
From Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools (2024) · cites this paper as [25]
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- Authors
- Thuronyi, B. W., Koblan, L. W., Levy, J. M., Yeh, W.-H., Zheng, C., Newby, G. A., Wilson, C., Bhaumik, M., Shubina-Oleinik, O., Holt, J. R., & Liu, D. R.
- Affiliations
- Broad Institute (US)
- Harvard University (US)
- Boston Children's Hospital (US)
- Corresponding author
- Liu, David R.
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2019-07-22
- Volume
- 37
- Issue
- 9
- Pages
- 1070-1079
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC6728210
- OpenAlex
- W2963629338
- Cited by
- 347
- References
- 65
- Citations by year
- 2026: 31 · 2025: 56 · 2024: 55 · 2023: 51 · 2022: 46 · 2021: 45 · 2020: 53 · 2019: 9
- FWCI
- 12.65
- Type
- journal article
- Language
- en
- Update notices
- correction (2019-08-12)
- 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)
- Funders
- U.S. Department of Health & Human Services | NIH | 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)
- United States Department of Defense | Defense Advanced Research Projects Agency (HR0011-17-2-0049)
- 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
- base editors, phage-assisted continuous evolution, cytosine base editors, editing efficiency, deaminase activity, by-product formation
- MeSH terms
- Gene Editing, Adenosine Deaminase, Animals, Base Sequence, Cell Line, Humans, Gene Expression Regulation, Enzymologic, Gene Targeting, Directed Molecular Evolution, Mice, INDEL Mutation, CRISPR-Cas Systems
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