Chimeric Meganucleases for Precise Genome Editing
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Solution Overview
Problem
Current genome engineering techniques face challenges in achieving highly specific DNA double strand breaks, leading to off-target effects and reduced efficiency in gene modification, particularly in introducing targeted integration, deletion, or mutation of polynucleotides.
Innovation Solution
Development of optimized endonucleases from the LAGLIDADG family with specific mutations and fusions, such as with zinc finger domains or TAL effector repeat units, to enhance specificity and activity, allowing for precise DNA recognition and cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If natural meganucleases are used for genome modification, then site-specific DNA cleavage is achieved, but the recognition sequence must be conserved or pre-engineered limiting applicability
Solution Approach 1:
The patent combines a DNA-binding domain (such as zinc finger or TAL effector domains) with a nuclease domain (FokI) to create a chimeric meganuclease. This merging allows the DNA-binding domain to provide customizable sequence recognition while the nuclease domain provides cleavage activity, thereby achieving both site-specificity and versatility across different genomes.
Solution Approach 2:
The invention creates composite enzymatic structures by fusing different functional domains (DNA-binding domain + nuclease domain) into a single chimeric protein. This composite structure integrates the sequence recognition capability of zinc fingers or TAL effectors with the cleavage capability of FokI nuclease, enabling customized genome editing with high specificity and broad applicability.
2Adaptability or versatility
If meganucleases are engineered with new DNA binding specificities, then customization to existing genome sites is achieved, but cleavage activity decreases
Solution Approach 1:
The invention divides the meganuclease into two separate functional modules: a DNA-binding domain (zinc finger or TAL effector) that can be customized for different target sequences, and a nuclease domain (FokI) that retains optimized cleavage activity. This segmentation allows independent optimization of each domain's function.
Solution Approach 2:
The FokI nuclease domain acts as an intermediary that bridges the DNA-binding domain and the cleavage function. By using FokI as the nuclease component, the invention maintains high cleavage activity while allowing the DNA-binding domain to be customized for different target sites through modular assembly.
3Adaptability or versatility
If meganucleases recognize similar sequences, then binding flexibility is achieved, but off-target effects increase
Solution Approach 1:
The invention applies local quality by designing the DNA-binding domain to recognize a specific target sequence with high precision at the binding site, while the FokI nuclease domain provides localized cleavage activity only when properly positioned. This ensures that cleavage occurs only at the intended target site with minimal off-target effects.
Solution Approach 2:
The invention replaces the natural meganuclease's single integrated recognition-cleavage mechanism with a modular system where the DNA-binding domain (zinc finger or TAL effector) provides sequence-specific binding and the FokI nuclease domain provides controlled cleavage. This substitution allows for more precise control over binding specificity and cleavage location, reducing off-target effects.
Data Source
AI summary
Provided are optimized endonucleases, as well as methods of targeted integration, targeted deletion or targeted mutation of [polynucleotides using optimized endonucleases.