Genome Editing with Single-Stranded Nucleic Acids
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Solution Overview
Problem
Current genome editing methods, such as those using ZFNs, suffer from low efficiency and high screening requirements due to the low rate of spontaneous recombination in cells, hindering rapid and accurate targeted genome editing.
Innovation Solution
A method involving the introduction of targeting endonucleases and single-stranded nucleic acids into cells to create a double-stranded break at a specific chromosomal site, followed by homology-directed repair using single-stranded nucleic acids for precise editing, including insertion, deletion, or modification of chromosomal sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If homologous recombination is used for targeted genome editing, then specificity is improved, but editing efficiency deteriorates due to low spontaneous recombination rates
Solution Approach 1:
The patent introduces single-stranded nucleic acid templates in advance before inducing double-strand breaks. This preliminary provision of repair templates ensures that when breaks occur (via ZFNs, TALENs, or CRISPR-Cas9), the homology-directed repair can immediately proceed with high efficiency using the pre-positioned templates, rather than relying on sparse spontaneous recombination events.
Solution Approach 2:
The patent changes the physical state and concentration parameters of nucleic acid templates. By using single-stranded nucleic acids at optimized concentrations and designing them with specific homology arm lengths, the patent creates optimal conditions for homology-directed repair, dramatically increasing editing efficiency while maintaining the specificity of targeted recombination.
2Ease of manufacture
If double-stranded circular DNA is used for editing, then integration capability is improved, but editing frequency deteriorates to only 1%
Solution Approach 1:
The patent fundamentally changes the nucleic acid template parameters from double-stranded circular DNA to single-stranded linear nucleic acids with specific homology arms. This parameter change transforms the repair mechanism from inefficient random integration to efficient homology-directed repair, achieving editing frequencies up to 70% while maintaining integration capability through the designed homology regions.
Solution Approach 2:
The patent segments the nucleic acid template into distinct functional regions: homology arms (for targeted integration) and cargo sequences (for the desired edit). This segmentation allows the template to be precisely positioned at the target locus through homology-directed repair, dramatically improving editing frequency compared to undifferentiated circular DNA.
3Measurement precision
If screening effort is increased to isolate targeted events, then accuracy is improved, but time and resource requirements deteriorate
Solution Approach 1:
The patent changes the frequency parameter of targeted events by using single-stranded nucleic acid templates with optimized homology arms and inducing controlled double-strand breaks. This increases targeted editing events from rare (1%) to common (up to 70%), eliminating the need for extensive screening and enabling direct identification of edited cells through simple markers or phenotypic selection.
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 enhances the speed and accuracy of genome editing, allowing for high-frequency targeted modifications, including the integration of specific sequences and deletions, as demonstrated by successful integration of sites like BamHI and HindIII into various loci across different cell types.
Implementation Method 1
at least one targeting endonuclease or nucleic acid encoding a targeting endonuclease, the targeting endonuclease being able to introduce a double-stranded break at a targeted cleavage site in the chromosomal sequence
Implementation Method 2
the double-stranded break introduced by the targeting endonuclease is repaired by a homology-directed process such that the chromosomal sequence is exchanged with the sequence of the single-stranded nucleic acid
Data Source
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AI summary
The present invention provides methods and kits for editing specific chromosomal sequences in cells. In particular, targeting endonucleases and single-stranded nucleic acids are used to edit the chromosomal sequence.