Single-Stranded Oligonucleotide Assembly for Gene Editing
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Solution Overview
Problem
Current gene editing techniques, such as CRISPR/Cas, face challenges in simplifying the assembly and delivery of donor nucleic acid molecules and guide-polynucleotides, with inefficiencies in integration and accuracy, particularly due to high rates of indel mutations at conjunction sites.
Innovation Solution
The use of at least two single-stranded oligonucleotides that are 80% complementary to each other for the assembly of double-stranded nucleic acid molecules into a single construct within a cell, facilitating the integration of these molecules into a predetermined sequence, thereby simplifying the assembly and delivery process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If single-stranded oligonucleotides are used as donor templates in CRISPR-Cas9-mediated genome editing, then the simplicity of delivery is improved, but the accuracy of integration deteriorates due to high rates of indel mutations at conjunction sites
Solution Approach 1:
The patent segments the donor nucleic acid into multiple overlapping single-stranded oligonucleotides (typically 3-5 oligos of 80-200 bp each) that are introduced separately into the cell. These oligonucleotides overlap by 20-50 bp at their junctions, allowing the cell's homologous recombination machinery to assemble them into a complete double-stranded donor template with high precision, thereby reducing indel mutations while maintaining delivery simplicity
Solution Approach 2:
The patent performs preliminary design and synthesis of multiple overlapping single-stranded oligonucleotides with optimized parameters (length, overlap regions, sequence composition) before introduction into the cell. This preliminary preparation ensures that the oligonucleotides are pre-configured for efficient assembly and high-fidelity integration, improving accuracy before the actual genome editing process begins
2Manufacturing precision
If multiple overlapping single-stranded oligonucleotides are assembled in vitro before transformation, then the assembly precision is improved, but the complexity of the process increases
Solution Approach 1:
The patent enables the oligonucleotides to self-assemble into double-stranded DNA structures within the cell using the cell's own homologous recombination machinery. This self-service approach eliminates the need for complex in vitro assembly procedures (such as PCR-based assembly or ligase reactions), reducing process complexity while maintaining high assembly precision through the cell's native repair mechanisms
Solution Approach 2:
The patent uses the cell's homologous recombination machinery as an intermediary to assemble the overlapping single-stranded oligonucleotides. This biological mediator automatically aligns and joins the oligonucleotides based on their overlapping sequences, providing precise assembly without requiring complex external assembly protocols or additional reagents
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 method enhances the efficiency and accuracy of nucleic acid integration, reducing indel mutations and improving the overall process of gene editing by allowing for the precise assembly of double-stranded nucleic acid constructs within cells.
Implementation Method 1
wherein the first and second single-stranded oligonucleotide are at least 80% complementary to each other over the whole sequence
Data Source
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AI summary
The present invention is based on the adavantageous use of single-stranded oligonucleotides in the in vivo (within a cell) assembly of double-stranded oligonucleotides into a single double-stranded nucleic acid construct. The present invention relates to the use of at least a first and a second single-stranded oligonucleotide in the assembly within a cell of at least two double-stranded nucleic acid molecules into a single double-stranded nucleic acid construct of pre-determined sequence, wherein the first and second single-stranded oligonucleotide are essentially complementary to each other.