CRISPR Editing with Heterologous DNA Repair Enzymes
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Solution Overview
Problem
Current CRISPR-Cas9 genome editing technologies face inefficiencies and off-target effects, limiting their development for therapeutic applications in humans.
Innovation Solution
Combining the Cas9 enzyme with guide RNA and heterologous DNA repair enzymes like RecBCD, AddAB, or AdnAB, which can be introduced into cells using expression vectors, to enhance DNA editing specificity and efficiency, particularly through homology-directed repair.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Cas9 enzyme is used for genome editing, then editing capability is achieved, but editing efficiency is sub-optimal and off-target effects occur
Solution Approach 1:
The patent introduces heterologous DNA repair enzymes (RecBCD, AddAB, or AdnAB) as intermediary components that work alongside Cas9 to enhance the editing process. These repair enzymes act as mediators that improve homology-directed repair efficiency, thereby increasing overall editing reliability while reducing off-target effects through more precise repair mechanisms
Solution Approach 2:
The patent creates a composite genome editing system by combining Cas9 enzyme with heterologous DNA repair enzymes in a single therapeutic composition. This composite approach integrates the gene-editing capability of Cas9 with the enhanced repair capabilities of the heterologous enzymes, achieving both high editing efficiency and reduced off-target effects
2Adaptability or versatility
If CRISPR-Cas9 system is used for therapeutic applications, then genetic disease treatment is enabled, but off-target effects remain a major obstacle
Solution Approach 1:
The heterologous DNA repair enzymes serve as intermediary components that bridge the gap between Cas9 cutting activity and precise genome repair. These intermediaries enhance the fidelity of the therapeutic application by ensuring more accurate homology-directed repair, thereby reducing off-target effects while maintaining therapeutic versatility
Solution Approach 2:
The patent changes the biochemical parameters of the genome editing system by introducing heterologous repair enzymes with different kinetic and specificity properties. This parameter change optimizes the repair process to favor on-target corrections while minimizing off-target modifications, enabling safer therapeutic applications
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 improves the precision and efficiency of CRISPR-based editing, reducing off-target effects and enhancing the ability to introduce specific mutations, thereby facilitating more effective genome editing therapies.
Implementation Method 1
Combining the Cas9 enzyme with guide RNA and heterologous DNA repair enzymes like RecBCD, AddAB, or AdnAB, which can be introduced into cells using expression vectors, to enhance DNA editing specificity and efficiency, particularly through homology-directed repair.
Data Source
AI summary
Provided are compositions, methods, and kits for improving CRISPR-based editing of DNA targets by a CRISPR-associated (Cas) enzyme. The improvement is made by combining the Cas enzyme and a CRISPR targeting RNA a heterologous DNA repair enzyme that is at least one of RecBCD, AddAB, or AdnAB. The heterologous DNA repair enzyme may have inactivated nuclease activity. The method can include using a DNA repair template to introduce one or more changes into the edited DNA. Cells that contain components of the improved CRISPR systems are included, as are kits for making such cells.