Transposase-Binding Nucleic Acid Complex for Size-Independent Genome Editing
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Solution Overview
Problem
Existing genome editing technologies, such as CRISPR/Cas9 and CRISPR-encoded transposase systems, are limited in their ability to perform complete exon recombination and are size-dependent, lacking user-controlled, single-strand or double-strand replacement capabilities.
Innovation Solution
A molecular complex comprising two single-stranded nucleic acid molecules with specific A/T-rich and G/C-rich sequences, oriented transposase recognition sites, and complementary base pairing to enable site-specific recombination and sequence replacement without size limitations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Difficulty of detecting and measuring
If CRISPR/Cas9 system is used for genome editing, then targeted gene editing capability is improved, but complete exon recombination capability deteriorates
Solution Approach 1:
The patent combines the CRISPR/Cas9 system with a transposase system into a unified genome editing tool. The Cas9 protein creates a double-strand break at the target site, while the transposase simultaneously facilitates the insertion of the desired sequence, enabling both targeted editing and complete exon recombination in a single system
Solution Approach 2:
The engineered system performs multiple functions: it can create double-strand breaks (Cas9 activity), facilitate sequence insertion (transposase activity), and enable complete exon recombination. This multi-functional approach allows the same system to address various genome editing needs that previously required different tools
2Ease of manufacture
If CRISPR-encoded transposase system is used, then integration capability is improved, but recombination process completeness deteriorates
Solution Approach 1:
The patent merges the CRISPR targeting mechanism with the transposase integration mechanism. The Cas9-gRNA complex guides the system to the precise target location, while the transposase executes the integration of the desired sequence, ensuring both accurate targeting and complete recombination in a coordinated manner
3Adaptability or versatility
If Prime Editing is used, then genetic replacement capability is improved, but size dependency limitation deteriorates
Solution Approach 1:
The system utilizes the cell's own DNA repair mechanisms (homologous recombination and non-homologous end joining) to complete the genome editing process. After Cas9 creates the double-strand break and the transposase inserts the desired sequence, the cell's intrinsic repair pathways finalize the integration, eliminating the need for the editor to directly manipulate the entire replacement sequence
4Productivity
If conventional genome editing tools are used, then editing efficiency is improved, but user control over single-strand or double-strand replacement deteriorates
Solution Approach 1:
The system is designed to be dynamically controllable by the user. By adjusting the design of the donor template and the choice of transposase system, users can control whether single-strand or double-strand replacement occurs. The system adapts its behavior based on the provided templates and cellular conditions, allowing flexible control over the editing outcome
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 controlled and efficient genome editing by guiding recombinases to specific sites for sequence replacement, applicable to any target sequence, independent of its nature, and utilizing the recombination properties of transposases.
Implementation Method 1
the first and second single-stranded nucleic acid molecules are paired according to the base complementarity defined by Watson and Crick so as to define two double-stranded binding sites for said transposase
Implementation Method 2
said first molecule comprising at its 5' end a first 5'-3' oriented transposase recognition sequence and at its 3' end at least one second transposase recognition sequence
Data Source
Figure 1~4
Figure 5~8
Figure 9A~9C
AI summary
The invention relates to a molecular complex that contains: - a first single-stranded nucleic acid molecule comprising at least two binding half-sites of a transposase; and - a second single-stranded nucleic acid molecule comprising at least one binding half-site of a transposase. The complex is such that the first and second single-stranded nucleic acid molecules are paired according to the base complementarity defined by Watson and Crick so as to define two double-stranded binding sites of said transposase. The invention further relates to the use of said complex, in particular for DNA editing.