Continuous evolution of base editors with expanded target compatibility and improved activity

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...

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Bibliographic Details
Published inNature biotechnology Vol. 37; no. 9; pp. 1070 - 1079
Main Authors Thuronyi, B W., Koblan, Luke W., Levy, Jonathan M., Yeh, Wei-Hsi, Zheng, Christine, Newby, Gregory A., Wilson, Christopher, Bhaumik, Mantu, Shubina-Oleinik, Olga, Holt, Jeffrey R., Liu, David R.
Format Journal Article
LanguageEnglish
Published New York Nature Publishing Group US 01.09.2019
Nature Publishing Group
Subjects
631/1647
631/1647/1511
Adenosine Deaminase - genetics
Adenosine Deaminase - metabolism
Agriculture
Animals
Base Sequence
Bioinformatics
Biomedical Engineering/Biotechnology
Biomedicine
Biotechnology
Cell Line
Compatibility
Context
CRISPR
CRISPR-Cas Systems
Cytosine
Directed Molecular Evolution
DNA sequencing
Editing
Efficiency
Evolution
Gene Editing
Gene Expression Regulation, Enzymologic
Gene Targeting
Humans
INDEL Mutation
Life Sciences
Methods
Mice
Mutation
Nucleotide sequencing
Phages
United States
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Summary: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. Improved base editors are generated by continuous evolution.
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Present address: B.W.T.: Department of Chemistry, Williams College, Williamstown, Massachusetts, USA.
B.W.T. designed the research, designed and constructed plasmids, and performed PACE and bacterial experiments. J.M.L. and L.W.K. designed and performed HEK cell experiments and analyzed data. J.M.L. designed and performed APOE editing experiments. W-H.Y. designed and performed baringo editing experiments. C.Z. constructed plasmids and performed bacterial experiments for selection development. G.A.N. designed and constructed plasmids for the HEK cell experiment for WFS1 editing. C.W. designed and performed ancestral sequence reconstruction. M.B., O.S-O., and J.R.H. contributed baringo mouse cells. D.R.L. designed and supervised the research. B.W.T., L.W.K., and D.R.L. wrote the manuscript. All authors contributed to editing the manuscript.
Author Contributions
These authors contributed equally
ISSN:1087-0156
1546-1696
1546-1696
DOI:10.1038/s41587-019-0193-0