High-diversity base mutagenesis via simultaneous adenine, cytosine and guanine editing

Development and Characterization of RNA Aptamer-Mediated Modular Base Editors Containing Staphylococcus aureus Cas9 Derivatives and Novel Deaminase Orthologs

The effectiveness of the SaCas9 modular base editors, the robustness of the platform’s modularity, and its feasibility for convenient screening of target-specific base editors are demonstrated.

A self-iterative orthogonal base-editing platform enables multiplex N-to-N diversification and genome-scale functional screening in Escherichia coli

Abstract 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.

From Double-Strand Breaks to Precision Edits: A Comparative Review of Modern Genome Editing Tools

This review compares Cas9-mediated homology-directed repair (HDR) with generations of cytosine base editors (CBE1–CBE3), adenine base editors (ABE1-ABE7), and prime editors (PE1–PE3b), focusing on their mechanistic distinctions, efficiencies, delivery challenges, and therapeutic applications.

Donor-complementary prime editing enables precise kilobase and library-compatible DNA insertions.

Recent advances in base editing technology and its applications in disease therapy.

This review focuses on the action mode of different base editors, highlights their interplays with the TLS, summarizes their potential therapeutic applications and discusses perspective strategies to improve precision and expand targeting scope.

A dual A-to-Y and G-to-Y base editor and a four-base concurrent hypermutator in mammalian cells

Current dual base editors and hypermutators exhibit limited types of base conversion and constrained mutational diversity. To address this challenge, we engineer a compact dual base editor “A&GBE” by fusing a deaminase and glycosylase with nickase Cas9 (nCas9) to enable concurrent adenine and cytosine editing. Furthermore, we develop quadruple base mutating modules (QBMM) by fusing nickase or dead Cas9 with engineered thymine-DNA glycosylase (TDG), N-methylpurine DNA glycosylase (MPG) and TadA8e, together with MS2-MCP system recruited activation-induced cytidine deaminase (AID). This QBMM enables simultaneous mutation of all four types of nucleotide bases (A, T, G, C) within an approximately 100 base pair (bp) sequence surrounding the gRNA target site. This platform generates hyper diverse multi-nucleotide variants, establishing a powerful tool for accelerated protein evolution, functional genomics, and disease modeling.