Endogenous DNA Editing Complex for Cytotoxicity Reduction
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Solution Overview
Problem
Existing genome editing techniques require the introduction of exogenous DNA modifying enzymes, leading to cytotoxicity and delivery issues due to the use of these enzymes.
Innovation Solution
A method utilizing a complex of a nucleic acid sequence-recognizing module and a DNA modifying enzyme-binding module that binds to cell-endogenous enzymes, allowing for DNA editing without exogenous enzymes, by stimulating the cell with a factor inducing the endogenous enzyme and contacting the complex with the DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If exogenous DNA modifying enzymes are introduced into the cell for genome editing, then DNA editing capability is achieved, but cytotoxicity and delivery issues occur
Solution Approach 1:
The invention utilizes the cell's own endogenous DNA modifying enzymes (such as APOBEC3G, AID, or AID2) to perform the DNA editing function. Instead of introducing external enzymes, the system activates the cell's intrinsic enzymatic machinery through inducible expression or activation mechanisms, thereby achieving DNA editing while avoiding the cytotoxicity and delivery problems associated with exogenous enzyme introduction
Solution Approach 2:
The invention employs an intermediary mechanism (such as guide RNA or protein adapters) that bridges the nucleic acid sequence recognition function and the endogenous enzyme activation function. This intermediary allows the system to target specific DNA sequences and recruit or activate endogenous enzymes at the target site without requiring direct introduction of the enzymes themselves into the cell
2Manufacturing precision
If exogenous DNA modifying enzymes are used, then targeted DNA conversion is achieved, but delivery efficiency is reduced
Solution Approach 1:
By leveraging the cell's endogenous enzymes that are already present or can be easily induced within the cell, the invention eliminates the need for complex delivery systems required for exogenous enzymes. The cell naturally produces or can be stimulated to produce these enzymes, thereby achieving high delivery efficiency while maintaining targeted DNA conversion capability through sequence-specific recognition modules
3Reliability
If endogenous DNA modifying enzymes are activated, then off-target actions are minimized, but control of enzyme activity is required
Solution Approach 1:
The invention achieves spatial and temporal control of endogenous enzyme activation by coupling enzyme activation or recruitment to sequence-specific recognition modules (such as CRISPR-Cas systems or zinc finger proteins). This ensures that the endogenous enzymes are activated or recruited only at the specific target DNA sequence location, minimizing off-target actions while maintaining simple control through the inherent specificity of the recognition modules
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
This approach reduces side effects and improves delivery efficiency by using cell-endogenous enzymes, allowing for controlled activity and minimizing off-target actions.
Implementation Method 1
by using deaminase that catalyzes a deamination reaction and by introducing a complex of the deaminase linked to a molecule having a DNA sequence recognition ability into the host cell
Data Source
AI summary
Provided is a method for altering a targeted site of a DNA in a cell, including a step of stimulating the cell with a factor inducing a DNA modifying enzyme endogenous to the cell, and bringing a complex of a nucleic acid sequence-recognizing module specifically binding to a target nucleotide sequence in a given DNA and a DNA modifying enzyme-binding module bonded to each other into contact with the DNA to convert one or more nucleotides in the targeted site to other one or more nucleotides or delete one or more nucleotides, or insert one or more nucleotides into the targeted site.