Donor DNA Template Composition for Higher HDR Genome Insertion
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Solution Overview
Problem
Current genome engineering tools lack the ability to control the division of labor between cellular DNA repair mechanisms, leading to stochastic repair processes that reduce the efficiency and accuracy of genome editing, particularly in cases requiring precise insertion of a DNA element at a break site.
Innovation Solution
A donor DNA template containing a transcription factor binding site is introduced to enhance the import of the template into the nucleus, promoting elevated rates of homology-directed repair (HDR) by leveraging transcription factors to bias the repair pathway.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current genome editing tools (ZFNs, TALENs, CRISPR/Cas) are used to generate DNA breaks, then sequence-specific DNA breaks are produced, but the repair is stochastic and predominantly favors NHEJ over HDR, reducing efficiency of donor DNA insertion
Solution Approach 1:
The patent introduces a mediator protein that specifically binds to the donor DNA template and facilitates its recruitment to the DNA break site. This mediator acts as an intermediary between the donor DNA and the HDR machinery, enhancing the efficiency of donor DNA insertion while promoting accurate HDR repair over stochastic NHEJ
Solution Approach 2:
The patent modifies parameters of the donor DNA template, such as adding specific sequences or structural features that increase its affinity for HDR machinery or its stability at the break site. These parameter changes shift the balance from NHEJ-dominated repair to HDR-favored repair, improving both efficiency and accuracy of donor DNA insertion
2Reliability
If NHEJ is the dominant repair process, then DNA breaks are repaired quickly, but the original sequence is frequently mutated, reducing accuracy of genome editing
Solution Approach 1:
The patent employs preliminary action by pre-processing the donor DNA template with specific modifications or by pre-assembling protein-DNA complexes that are primed for HDR. This preliminary preparation ensures that when the DNA break occurs, the HDR machinery is already positioned and ready to act, reducing the time loss associated with stochastic repair selection while maintaining high accuracy
3Productivity
If donor DNA template is delivered to cells without enhancement, then the process is simple, but the import into the nucleus and HDR repair rate is low
Solution Approach 1:
The patent uses a mediator protein as an intermediary carrier that binds to the donor DNA template and facilitates its nuclear import. This mediator simplifies the delivery process by providing a single functional unit (protein-DNA complex) that can be efficiently transported into the nucleus, thereby increasing HDR repair rate without significantly complicating the delivery mechanism
Solution Approach 2:
The patent creates a composite structure by combining the donor DNA template with a mediator protein to form a functional complex. This composite material has enhanced properties for nuclear import and HDR engagement compared to naked donor DNA, achieving higher repair rates while maintaining relatively simple delivery protocols
Data Source
AI summary
The present invention relates to compositions and methods for increasing the rate of site specific insertion of a donor DNA sequence to the genome. More specifically, the method introduces a donor DNA template containing at least one transcription factor binding site to a cell in order to favor specific insertion of a donor template sequence at a target site by homology directed repair (HDR).