Split Deaminase Base Editor for Controlled Editing
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Solution Overview
Problem
Current base editors face limitations due to naturally constrained deaminase activity and increased oncogenic mutations, as well as undesirable off-target effects, which restrict their broader application in gene therapy and therapeutic utility.
Innovation Solution
Development of split DNA deaminase encoding constructs that can be reconstituted at specific sites using small molecule dimerization agents, allowing for precise and controlled base editing with reduced off-target activity by incorporating targeting and modifying modules that localize and edit DNA sequences efficiently.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DNA deaminase is overexpressed to enhance base editing activity, then editing efficiency is improved, but off-target effects and oncogenic mutations increase
Solution Approach 1:
The DNA deaminase is divided into two separate polypeptide fragments (first fragment and second fragment) that cannot function independently. Only when both fragments are present and interact do they reconstitute the active deaminase, enabling controlled editing activity while preventing off-target effects from overexpressed active enzyme.
Solution Approach 2:
A dimerization agent acts as an intermediary to induce the association of the two deaminase fragments. This external mediator provides temporal and spatial control over enzyme reconstitution, allowing editing activity to be activated only when and where needed, thereby reducing unwanted off-target activity.
2Measurement precision
If DNA deaminase is targeted to specific genomic loci, then site-specific editing is achieved, but the enzyme remains active and accessible to non-target ssDNA intermediates
Solution Approach 1:
The deaminase is segmented into inactive fragments that are delivered together with the targeted dCas9-sgRNA complex. The fragments remain inactive until they reconstitute upon association with the targeting module, ensuring that even if the complex binds to non-target sites, the deaminase activity is not present to cause off-target mutations.
Solution Approach 2:
The targeting module (dCas9-sgRNA) is assembled and delivered to the target site before the deaminase fragments become active. This preliminary positioning ensures that when the fragments reconstitute, the active enzyme is already localized at the intended target site, preventing genome-wide mutations.
3Productivity
If deaminase activity is enhanced to overcome natural constraints, then base editing capability is improved, but catalytic hyperactivation leads to increased oncogenic mutations
Solution Approach 1:
The deaminase is divided into fragments that must reconstitute to become active. This segmentation allows the use of deaminases with enhanced catalytic activity without the risk of hyperactivation, because the active enzyme is only formed when both fragments are present and properly assembled, providing an inherent safety mechanism against oncogenic mutations.
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
Enables precise and controllable site-specific editing with enhanced specificity and reduced off-target effects, improving the therapeutic potential of base editors by allowing for targeted gene editing with minimal unwanted mutations.
Implementation Method 1
specific binding pair members dimerize upon contact with a dimerization agent causing two portions of the split deaminase enzyme to reform
Data Source
AI summary
Compositions and methods for small molecule control of precise base editing are disclosed.