Nuclease-Mediated Nucleic Acid Insertion via Segmented Oligonucleotides
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Solution Overview
Problem
Current methods for inserting desired nucleic acids into cells are inefficient, particularly in organisms with low homologous recombination efficiency, and often require complex vector production or result in inaccurate insertion of short DNA sequences.
Innovation Solution
A method involving a nuclease that specifically cleaves regions flanking a predetermined site in a nucleic acid, allowing for microhomology-mediated end joining (MMEJ) to accurately insert a desired nucleic acid of several kilobases in length, using a vector with sequences matching the cleavage sites and a nuclease including a homodimeric DNA cleavage domain or RNA-guided nuclease like CRISPR/Cas.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If homologous recombination is used to insert foreign DNA into cells, then insertion can be achieved, but the vector required is long-stranded and cannot be easily produced, and efficiency is low in certain cells and organisms
Solution Approach 1:
The donor DNA is segmented into two separate oligonucleotides (first oligonucleotide and second oligonucleotide) that each contain a portion of the desired sequence. These segmented oligonucleotides are easier to synthesize and handle than long-stranded vectors, while still enabling efficient insertion through the nuclease-mediated repair process.
Solution Approach 2:
A nuclease is introduced as an intermediary agent that creates double-strand breaks at specific sites flanking the insertion target. This nuclease-mediated break facilitates the insertion of the segmented oligonucleotides through cellular repair mechanisms, bypassing the need for complex long-stranded vectors while maintaining high efficiency.
2Length of moving object
If ssODN-mediated gene modification is used to introduce foreign DNA into animal embryos, then insertion can be achieved, but only short DNA sequences of about several 10 bp can be introduced
Solution Approach 1:
The desired DNA sequence is divided into two separate oligonucleotides that can be synthesized at lengths exceeding 10 bp each. These segmented oligonucleotides are introduced into animal embryos where the nuclease creates breaks that facilitate their integration, enabling insertion of longer sequences than traditional ssODN methods while maintaining efficiency.
3Manufacturing precision
If non-homologous end joining is used to insert foreign DNA by cleaving nucleic acid and joining cleaved sites, then insertion can be achieved, but the direction of insertion cannot be controlled and junction accuracy is poor
Solution Approach 1:
The oligonucleotides are designed with specific local qualities: each contains a region complementary to the sequences flanking the insertion site (enabling directional alignment) and a region that will become part of the inserted sequence. This local differentiation ensures accurate junction formation and controlled insertion direction while simplifying the overall process.
4Reliability
If heterodimeric ZFNs and heterodimeric TALENs are used to prevent re-cleavage of inserted DNA, then direction control and accurate joining can be achieved, but highly-active homodimeric artificial nucleases cannot be used
Solution Approach 1:
Instead of using heterodimeric nucleases to prevent re-cleavage, the invention inverts the approach by designing oligonucleotides with sequences that, when inserted, eliminate the nuclease recognition sites. This allows the use of highly-active homodimeric nucleases for the initial cut while ensuring the inserted DNA cannot be re-cleaved, thus achieving both high activity and insertion stability.
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
Enables high-frequency, accurate insertion of relatively long nucleic acids into various organisms without requiring complex vector production, maintaining stability and allowing for precise junction design, suitable for both undifferentiated cells and animal embryos.
Implementation Method 1
the nuclease specifically cleaves a moiety including the region formed of the first nucleotide sequence and the region formed of the second nucleotide sequence included in the cell
Implementation Method 2
the first nucleotide sequence in the nucleic acid in the cell and the first nucleotide sequence in the vector are joined by microhomology-mediated end joining (MMEJ)
Data Source
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AI summary
The present invention provides the following: a vector for inserting a desired nucleic acid into a predetermined site of a nucleic acid comprising a region formed of a first nucleotide sequence, the predetermined site, and a region composed of a second nucleotide sequence, in the stated order in the 5'-to-3' direction, wherein the vector comprises a region formed of the first nucleotide sequence, the desired nucleic acid, and the second nucleotide sequence in the stated order in the 5'-to-3' direction; a kit that includes this vector; a method of inserting a nucleic acid comprising a step for introducing this vector into a cell; a cell acquired by this method; and an organism comprising this cell.