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Nat Biotechnol DOI:10.1038/s41587-019-0193-0

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

Publication TypeJournal Article
Year of Publication2019
AuthorsThuronyi, BW, Koblan, LW, Levy, JM, Yeh, W-H, Zheng, C, Newby, GA, Wilson, C, Bhaumik, M, Shubina-Oleinik, O, Holt, JR, Liu, DR
JournalNat Biotechnol
Volume37
Issue9
Pages1070-1079
Date Published2019 Sep
ISSN1546-1696
Abstract

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.

DOI10.1038/s41587-019-0193-0
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/31332326?dopt=Abstract

Alternate JournalNat. Biotechnol.
PubMed ID31332326
PubMed Central IDPMC6728210
Grant List / HHMI / Howard Hughes Medical Institute / United States
U01 AI142756 / AI / NIAID NIH HHS / United States
R01 DC013521 / DC / NIDCD NIH HHS / United States
HR0011-17-2-0049 / / United States Department of Defense | Defense Advanced Research Projects Agency (DARPA) /
/ / Howard Hughes Medical Institute / United States
T32 GM095450 / GM / NIGMS NIH HHS / United States
R35 GM118062 / GM / NIGMS NIH HHS / United States
Liu Investigatorship / / Howard Hughes Medical Institute (HHMI) /
R01 EB022376 / EB / NIBIB NIH HHS / United States
RM1 HG009490 / HG / NHGRI NIH HHS / United States