Recombinant Nucleases with TALE Binding for Precise HDR Editing
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Solution Overview
Problem
Existing genome editing technologies, such as TALENs and CRISPR, face challenges in achieving precise and efficient genome modifications due to the predominance of the error-prone non-homologous end joining pathway in mammalian cells, limiting the effectiveness of homology-directed repair.
Innovation Solution
Development of recombinant nucleases operatively linked to nucleic acid binding domains, specifically TALE DBDs, which allow for targeted and precise genome editing by binding to specific DNA sequences and cleaving at defined sites, enabling efficient use of the HDR pathway.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If TALENs or CRISPR-mediated genome editing tools are used to introduce double-strand breaks, then genome editing efficiency is improved, but precision and accuracy deteriorate due to the error-prone NHEJ pathway
Solution Approach 1:
The patent introduces a donor DNA template as an intermediary element that mediates the repair process. Instead of relying solely on the error-prone NHEJ pathway, the donor DNA template serves as a blueprint for precise repair through HDR, enabling accurate genome editing while maintaining high efficiency
Solution Approach 2:
The patent changes the repair pathway parameter from NHEJ to HDR by providing a donor DNA template. This parameter change transforms the repair mechanism from error-prone to high-fidelity, thereby improving precision while maintaining the efficiency gains from DSB induction
2Productivity
If traditional genome editing tools are used, then double-strand breaks can be introduced, but targeting specificity and accuracy deteriorate
Solution Approach 1:
The patent segments the genome editing system into distinct functional modules: a nuclease domain for DSB introduction, a DNA binding domain for specific target recognition, and a donor DNA template for precise repair. This segmentation allows each component to be optimized independently, improving both productivity and targeting specificity
Solution Approach 2:
The patent creates a universal genome editing platform that combines multiple functions: the nuclease domain provides DSB capability, the DNA binding domain provides targeting specificity, and the donor template provides repair functionality. This multi-functional system resolves the contradiction between productivity and precision
3Productivity
If NHEJ pathway is used for repair, then genome editing can proceed efficiently, but accuracy and fidelity deteriorate
Solution Approach 1:
The donor DNA template acts as an intermediary that shifts the repair mechanism from NHEJ to HDR. This intermediary element provides the necessary information for accurate repair while maintaining the efficiency benefits of programmed DSBs
Solution Approach 2:
The patent converts the harmful effect of NHEJ (error-prone repair) into a benefit by using the DSB it creates as a entry point for HDR-mediated precise editing. The initial break, which could lead to errors, becomes the starting point for high-fidelity repair when a donor template is provided
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 recombinant nucleases achieve high efficiency and precision in genome editing, facilitating genetic modifications like knock-ins, knock-outs, and mutations, and maintaining or increasing CD8+ cell ratios in immune cells.
Implementation Method 1
the nucleic acid binding domain specifically binds to a target nucleic acid sequence in a nucleic acid molecule
Implementation Method 2
the cleavage domain introduces double-strand breaks (DSBs) in mammalian genome efficiently
Implementation Method 3
the HDR pathway allows for precise genome editing via the use of sister chromatids or exogenous DNA molecules
Data Source
AI summary
The present disclosure relates to recombinant nucleases, recombinant nucleases operatively linked to nucleic acid binding domains, and methods of making and using them.