Nucleic acids researchAug 10, 2026

A repeated base-editing method allows multiple targeted changes and large-scale function testing in E. coli

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Aug 11, 2026

Abstract

MUTATOR enabled the generation of 84 distinct amino-acid combinations from a four-site editing of ompR.

MUTATOR combines two base-editing methods to allow N-to-N diversification in Escherichia coli.
This platform overcomes limitations of traditional genome editing by expanding nucleotide outcomes and amino-acid diversity.
Four gRNAs facilitated the editing of ompR, resulting in 252 codon combinations.
The OmpR_P160P variant increased isobutanol production by up to 56.2%.
MUTATOR was applied to a library of 151 genes, uncovering mutations that improved cell growth and ethanol utilization.
These findings position MUTATOR as a versatile tool for genome-wide modifications and microbial engineering.

Simplified

Base editing enables precise genome modification without double-strand breaks but remains limited by narrow editing windows, DNA repair pathway biases, and restricted nucleotide diversity. Here, we report MUTATOR, a MUlTiplexAble and self-iTerative ORthogonal base-editing platform that enables N-to-N diversification in Escherichia coli. MUTATOR combines CWBE and ABE with iterative editing on two complementary DNA strands, thereby overcoming endogenous DNA repair constraints and expanding A-to-N and C-to-N editing outcomes across both strands. This strategy substantially expands accessible nucleotide outcomes, codon variants, and amino-acid diversity within existing editing windows relative to conventional editors. Using four gRNAs, MUTATOR facilitated four-site editing of ompR, generating 84 distinct amino-acid combinations and 252 codon combinations, with the synonymous OmpR_P160P variant increasing isobutanol production by up to 56.2%. We further applied MUTATOR to a 151-gene library encompassing transcriptional regulators, translation factors, DNA repair proteins, ribosomal components, and NAD(P)H-associated metabolic genes, identifying single and combinatorial mutations that markedly enhanced cell growth and ethanol utilization when ethanol was used as the sole carbon source. Together, these results establish MUTATOR as a broadly applicable platform for genome-wide diversification, functional dissection, and rapid engineering of industrial microbial chassis.

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A repeated base-editing method allows multiple targeted changes and large-scale function testing in E. coli

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