Transposase-Binding Nucleic Acid Complex for PAM-Independent Editing
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Solution Overview
Problem
Existing genome editing technologies, such as CRISPR/Cas9 and Prime Editing, are limited by the size of the sequence they can manipulate and lack user-controlled, site-specific recombination capabilities.
Innovation Solution
A molecular complex comprising two single-stranded nucleic acid molecules with specific sequences that allow for controlled recombination and replacement of DNA strands, independent of sequence size, using transposases for targeted recombination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR/Cas9 is used for genome editing, then targeted gene modification is achieved, but the system is limited by sequence size and requires PAM-type sequences
Solution Approach 1:
The patent replaces the CRISPR/Cas9 molecular scissor mechanism with a transposase-based recombination system. Instead of using CRISPR guide RNA and Cas9 nuclease that require PAM sequences and have size limitations, the invention uses transposases with specific recognition sequences (ATT sites) that can manipulate sequences of any size without PAM requirements, thereby substituting one molecular mechanism for another more versatile one
Solution Approach 2:
The invention changes the fundamental parameters of the genome editing system by replacing the PAM-dependent CRISPR mechanism with a PAM-independent transposase system. The key parameter change is the recognition sequence requirement: from PAM sequences in CRISPR to ATT sites in transposase, which allows unlimited sequence size manipulation and removes the PAM constraint entirely
2Manufacturing precision
If Prime Editing is used for gene replacement, then single-stranded DNA replacement is achieved, but the system remains limited by cellular integration and repair mechanisms
Solution Approach 1:
The patent introduces dynamic control over the type of DNA replacement by using a system where the outcome (single-stranded or double-stranded replacement) can be controlled by the user. The transposase system allows dynamic adjustment of replacement parameters through the design of the nucleic acid molecule structure and transposase binding sites, enabling flexible switching between different replacement modes based on experimental needs
Solution Approach 2:
The invention creates a universal genome editing platform that can perform multiple functions: single-stranded replacement, double-stranded replacement, and controlled recombination. The transposase system serves as a multi-functional tool that can adapt to different editing requirements, unlike Prime Editing which is specialized for single-stranded replacement
3Manufacturing precision
If transposon Tn7 integration is used, then targeted integration is achieved, but the system does not provide true recombination
Solution Approach 1:
The patent merges the targeted integration capability of transposons with true recombination functionality. By combining transposase-mediated targeted integration with homology-directed repair pathways, the system achieves both precise targeting (like transposons) and genuine recombination (exchange of genetic material), creating a hybrid system that exhibits properties of both mechanisms
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 site-specific, user-controlled recombination and replacement of DNA sequences without reliance on PAM-type sequences, allowing for precise genome editing.
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 of said transposase
Data Source
AI summary
A molecular complex that contains: a first single-stranded nucleic acid molecule including at least two binding half-sites of a transposase, and a second single-stranded nucleic acid molecule including at least one binding half-site of a transposase. The complex is such that the first and second single-stranded nucleic acids are paired according to the base complementarity defined by Watson and Crick so as to define two double-stranded binding sites of said transposase. Also the use of the complex, in particular for DNA editing.