Single-Stranded DNA Templates for Large-Sequence Genome Editing
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Solution Overview
Problem
Current methods for inserting large DNA sequences into cells, such as those over 100 bases, are inefficient and laborious, particularly when using double-stranded DNA templates, while single-stranded DNA templates of similar length have not been effectively utilized due to synthesis limitations and incorporation uncertainties.
Innovation Solution
The use of single-stranded DNA (ssDNA) templates longer than 200 nucleotides, combined with relatively short homology arms, for targeted DNA modification in cells, facilitated by nuclease systems like CRISPR/Cas, enables efficient insertion of large DNA sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If double-stranded DNA templates are used for inserting large DNA sequences (>100 bases), then the insertion can be attempted, but the insertion efficiency is poor and the process is laborious
Solution Approach 1:
The patent changes the fundamental parameter of DNA template structure from double-stranded to single-stranded. This parameter change enables high-efficiency insertion of large DNA sequences (>100 bases) while simplifying the overall process, directly resolving the contradiction between insertion efficiency and ease of manufacture
2Productivity
If single-stranded DNA templates longer than 200 bases are used, then high insertion efficiencies are achieved, but synthesis limitations and incorporation uncertainties previously prevented effective utilization
Solution Approach 1:
The patent performs preliminary actions by optimizing ssDNA synthesis methods and designing appropriate homology arm lengths before the insertion process. This preliminary preparation eliminates synthesis limitations and incorporation uncertainties, enabling reliable use of long ssDNA templates with high insertion efficiency
Solution Approach 2:
The patent changes the DNA template from double-stranded to single-stranded form, which fundamentally alters the incorporation mechanism. This parameter change eliminates the uncertainties associated with dsDNA incorporation while maintaining high efficiency, directly resolving the reliability concern
3Length of moving object
If ssDNA repair templates with long DOIs (>100 bases) are used, then the overall length of ssDNA synthesis is limited and incorporation into target sequence is uncertain
Solution Approach 1:
The patent changes the structural parameter of the DNA template from double-stranded to single-stranded, which enables reliable incorporation of long DNA sequences (>100 bases). This parameter change directly addresses both the length limitation and incorporation reliability issues
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 achieves high insertion efficiencies, up to 100% overall and 50% in both alleles, demonstrating the effectiveness of ssDNA for modifying genomic DNA and altering gene expression.
Implementation Method 1
a nuclease system capable of cutting the target DNA sequence
Implementation Method 2
By introducing one of these targeted nuclease systems into a cell along with a repair template DNA encoding sequences or 'arms' homologous to the upstream and downstream sequences near the nuclease cut site, the HDR pathway can be used to insert a DNA of interest
Data Source
AI summary
Disclosed are compositions, methods, and kits for modifying DNA within cells as well as compositions and methods for modifying gene expression in a cell. In particular, the invention generally relates to compositions, methods, and kits for DNA editing using single-stranded DNA. Compositions and methods for modifying gene expression using artificial microRNAs (amiRNA) are also contemplated.