DNA Nanostructure Homology-Directed Repair Efficiency
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current gene-editing methods using CRISPR-Cas systems face challenges in efficiently integrating large DNA inserts via homology-directed repair (HDR), as long donor DNA molecules are prone to nuclease-mediated degradation and poor integration due to their linear structure.
Innovation Solution
The use of scaffolded DNA origami structures, where single-stranded donor DNA is folded into compact nanostructures using staple oligonucleotides, bringing homology arms into proximity, enhances the efficiency of HDR by protecting the DNA from degradation and facilitating precise integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If long donor DNA molecules are used for homology-directed repair, then the ability to integrate large DNA inserts is improved, but the DNA is prone to nuclease-mediated degradation and poor integration
Solution Approach 1:
The patent divides the long donor DNA molecule into multiple shorter oligonucleotide segments that are then assembled into a complete DNA insert within the HDR template. This segmentation protects the DNA from nuclease degradation while maintaining the ability to integrate large genetic material. Each oligonucleotide segment is individually stable and can be efficiently incorporated by the cellular repair machinery.
Solution Approach 2:
The patent employs a nested structure where multiple oligonucleotide segments are incorporated within a larger HDR template structure. The template contains homology arms that flank the insert region, creating a nested arrangement where the insert segments are protected within the context of the full template structure, enhancing both stability and integration efficiency.
2Ease of manufacture
If linear donor DNA structure is used, then simplicity of design is maintained, but integration efficiency and precision are reduced
Solution Approach 1:
The patent applies preliminary action by pre-assembling the donor DNA into a structured HDR template with defined homology arms and insert regions before introduction into the cell. The oligonucleotide segments are designed and synthesized with specific sequences that will be assembled into the final structure, allowing precise control over the integration outcome while maintaining design simplicity through modular construction.
Solution Approach 2:
The HDR template structure serves as an intermediary between the simple oligonucleotide segments and the complex integration process. This intermediate structure organizes the DNA segments with proper spacing and orientation, facilitating precise integration while keeping the overall design process simple and modular.
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 significantly improves the integration efficiency of large DNA inserts by creating a compact, nuclease-resistant DNA nanostructure that effectively brings homology arms into close proximity, enhancing the precision and efficacy of gene editing.
Implementation Method 1
The one or more staple oligonucleotides are least partially complementary to the donor DNA such that the one or more staple oligonucleotides hybridize to the donor DNA, such that the donor DNA folds into a nanostructure
Data Source
AI summary
The present disclosure provides compositions comprising a gene-editing polypeptide, a single-stranded donor DNA, and one or more staple oligonucleotides. The present disclosure provides compositions comprising a DNA nanostructure and a gene-editing polypeptide. The present disclosure provides gene editing methods using the compositions. The present disclosure provides methods of using the compositions to produce a genetically modified cell. The present disclosure provides kits useful for carrying out gene editing.