Context-Specific Cytosine Base Editors For Precise Single-Nucleotide DNA Editing

SUMMARY

Engineered TadA deaminase via directed evolution to produce CRISPR cytosine base editors that precisely convert target cytosines to thymines in specific DNA contexts with high efficiency and minimal off-target effects

The Unmet Need: Gene editing tools that exclusively acting on cytosine at levels comparable to naturally occurring deaminases reducing off-target effects

• Gene editing has revolutionized molecular biology and holds immense therapeutic promise. CRISPR-Cas systems enable programmable DNA targeting, and base editors fuse nucleotide deaminases to mediate direct conversion of C:G→T:A or A:T→G:C without double-strand breaks. Precise single-nucleotide resolution is essential for correcting point mutations associated with genetic disorders, many of which arise in contexts containing multiple identical bases. Unwanted edits at nearby nucleotides reduce safety and efficacy, underscoring the need for more context-specific editing tools.

• Despite successes, current base editors suffer from bystander edits, off-target activity, and context dependence. Many cytosine base editors indiscriminately deaminate all cytosines within a 4–5 nucleotide window, leading to unwanted mutations that complicate downstream analyses or therapeutic applications. Editing efficiency often varies dramatically with local sequence context, leaving many disease-relevant loci inaccessible, while off-target RNA and DNA deamination pose additional safety risks. The inability to selectively modify a single nucleotide among adjacent identical bases further limits precision and restricts the correction of pathogenic point mutations.

The Proposed Solution: Targeted strategy for altering the substrate specificity of E. coli TadA enabling the conversion of a dual adenine and cytosine deaminase into variants that selectively deaminate cytosine

• The faculty inventor harnessed directed evolution of the E. coli TadA deaminase through strategic targeting of three nucleic acid–recognition regions to reprogram substrate preference. Multiple rounds of targeted mutagenesis, bacterial selection, DNA shuffling and in vitro and cellular assays yielded a panel of context-specific cytosine base editors that convert C:G to T:A within designated NCN sequence contexts. These variants largely eliminate unwanted adenine deamination and bystander editing, delivering improved on-target efficiency and reduced off-target activity in mammalian cells.

• Existing cytosine base editors often suffer from bystander edits, limited single-nucleotide selectivity and variable efficiency across sequence contexts. By contrast, the evolved TadA-derived editors achieve precise conversion of a single cytosine among multiple. A panel of 16 NCN-preferring variants exhibits high editing efficiency (up to 68%) with minimal adenine editing (<0.4%) and broad correction of disease-associated sites offering unparalleled precision and expanded target scope for research and therapeutic applications.

ADVANTAGES

ADVANTAGES

• Precise C-to-T editing in specific NCN contexts, eliminating bystander and off-target A deamination

• High on-target efficiencies (up to ~68.7%) across a panel of 16 context-specific deaminases

• Minimal off-target activity in DNA and RNA (<0.4% unwanted A editing and reduced genome-wide/off-target edits)

• Enables precise correction of clinically relevant mutations (e.g., KRAS G12D, TP53 R248Q) in human cells

• Modular, evolvable platform for tailoring deaminases to any NCN context via bacterial selection

APPLICATIONS

• Drug development tool
• Target validation
• Gene therapy
• Personalized therapeutic editing
• Crop trait engineering
• Cell line creation

PUBLICATIONS

• Wu, Y., Xiao, YL. & Tang, W. High-precision cytosine base editors by evolving nucleic-acid-recognition hotspots in deaminase. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02678-w

June 12, 2025

Proof of concept

Patent Pending

Licensing,Co-development

Weixin Tang