Zinc Finger Base Editors Using TDD Dimerization for Precise C-to-T Editing

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Solution Overview

Problem

Existing DNA editing technologies, such as those using DddA-derived cytosine base editors, are limited in their applicability and efficiency for precise base editing in double-stranded DNA, particularly for treating genetic disorders.

Innovation Solution

Development of zinc finger protein (ZFP)-based nucleobase editing systems comprising fusion proteins with toxin-derived cytidine deaminase (TDD) domains that bind specifically to genomic regions, allowing for precise conversion of cytosine to thymine through dimerization and inclusion of nickase components to enhance editing efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If DddA-derived cytosine base editors are used for base editing in double-stranded DNA, then cytosine to thymine conversion can be achieved, but the applicability and efficiency are limited

Engineering Contradiction:

Improvebase editing efficiencyVSAvoidapplicability breadth

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The patent changes the parameters of the base editing system by using toxin-derived deaminases (TDDs) instead of DddA, and by creating fusion proteins with specific zinc finger domains that bind to particular DNA sequences. This results in improved editing efficiency and expanded applicability to different genomic locations and disease targets.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention creates composite fusion proteins combining zinc finger domains with toxin-derived deaminase domains. These composite proteins exhibit enhanced base editing activity and specificity compared to the original DddA system, resolving the contradiction between efficiency and adaptability.

Inventive Principle:

Principle #40Composite materials

2Manufacturing precision

If zinc finger protein fusion proteins with toxin-derived cytidine deaminase are used, then precise base conversion is achieved, but the system complexity increases

Engineering Contradiction:

Improvebase editing precisionVSAvoidsystem complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The base editing system is segmented into functional modules: zinc finger domains for DNA binding, toxin-derived deaminase domains for cytosine conversion, and optional nickase components for enhancing precision. This modular design achieves high precision while maintaining manageable complexity through standardized components.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The zinc finger fusion protein system is designed to be multi-functional and adaptable. The same basic architecture can target different genomic locations and treat various diseases by simply changing the zinc finger domain sequences, reducing the need for entirely new systems for each application.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Reliability

If dimerization of cytidine deaminase portions is used, then active enzyme formation is achieved, but the requirement for paired fusion proteins increases complexity

Engineering Contradiction:

Improveenzyme activity reliabilityVSAvoidprotein structure complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

Two separate fusion proteins are designed to dimerize and form an active cytidine deaminase enzyme. Each protein contains a zinc finger domain and a deaminase portion, and their pairing through dimerization creates the functional enzyme complex. This merging approach ensures reliable enzyme activity while using a systematic design that manages complexity.

Inventive Principle:

Principle #5Merging (Combining)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The ZFP-TDD systems enable precise and efficient conversion of C:G to T:A base pairs, facilitating the treatment of genetic disorders by allowing for simultaneous knock-out of multiple genes and improving editing efficiency compared to existing technologies.

Implementation Method 1

a first zinc finger protein (ZFP) domain that binds to a first sequence in a target genomic region

Methodology Applied

Scientific EffectSequence-specific DNA binding:

Implementation Method 2

Cytidine deaminases convert the nucleobase cytosine to thymine (or the nucleoside deoxycytidine to thymidine)... DddA can catalyze the deamination of cytosine to uracil within double-stranded DNA

Methodology Applied

Scientific EffectCytidine deamination: Enzyme

Implementation Method 3

binding of the first fusion protein and the second fusion protein to the target genomic region results in dimerization of the first and second portions

Methodology Applied

Scientific EffectProtein dimerization:

Data Source

PatentUS20250361528A1Novel zinc finger fusion proteins for nucleobase editing

Publication Date: 2025.11.27 SANGAMO THERAPEUTICS INC

AI summary

Provided herein are base editor systems comprising fusion proteins that comprise zinc finger protein and cytidine deaminase domains, as well as methods of using the base editor systems. The systems can be used to specifically alter a single base pair in a target DNA sequence.