DNA Polymerase Genome Editing for Precise Sequence Replacement
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Solution Overview
Problem
Current genome editing methods, such as homologous recombination-mediated repair and prime editing, suffer from low efficiency in precisely replacing target genome sequences, leading to potential mutations and inefficiencies in disease treatment and agronomic trait improvement.
Innovation Solution
A genome editing system utilizing a sequence-specific nuclease, DNA polymerase, and a single-stranded DNA template, optionally with a fusion protein or recruiting protein, to introduce precise modifications by forming a complex within a cell and extending the DNA sequence with a template.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If homologous recombination-mediated repair is used for precise genome editing, then target sequence replacement can be achieved, but editing efficiency is low and mutations may occur
Solution Approach 1:
The patent combines sequence-specific nuclease (for precise target recognition and cleavage) with DNA polymerase (for efficient and accurate DNA synthesis) into a unified genome editing system. This merging allows the system to simultaneously achieve precise target sequence replacement through nuclease-mediated cleavage and high editing efficiency through polymerase-mediated DNA repair, resolving the contradiction between precision and productivity in genome editing
Solution Approach 2:
The patent introduces a single-stranded DNA template as an intermediary component that mediates the transfer of precise sequence information from the external template to the target genome location. This template serves as a bridge between the nuclease-created double-strand break and the DNA polymerase-mediated repair process, ensuring accurate sequence replacement while maintaining high editing efficiency by guiding the repair mechanism
2Adaptability or versatility
If prime editing system with reverse transcriptase is used, then sequence rewriting can be achieved, but system complexity increases
Solution Approach 1:
The patent extracts the reverse transcriptase component from the prime editing system and replaces it with DNA polymerase. This extraction eliminates the need for complex pegRNA design and reverse transcription steps while retaining the core functionality of sequence rewriting. The simplified system uses only DNA-based components (nuclease, DNA polymerase, and single-stranded DNA template), reducing system complexity while maintaining adaptability for various sequence editing applications
Solution Approach 2:
The patent changes the fundamental parameter of the editing mechanism from RNA-based reverse transcription to DNA-based polymerization. By switching from reverse transcriptase (RNA-dependent DNA polymerase) to standard DNA polymerase (DNA-dependent DNA polymerase), the system achieves sequence rewriting capability through a simpler, more direct biochemical pathway, reducing complexity while preserving versatility
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
Enhances the precision and efficiency of genome editing by allowing targeted sequence replacements with reduced mutation rates, facilitating effective disease treatment and agronomic trait enhancements.
Implementation Method 1
the sequence-specific nuclease can specifically target (bind to) a target sequence and introduce a double-stranded break (DSB) or single-stranded nick (nick) in or near the target sequence
Implementation Method 2
a DNA polymerase... and a single-stranded DNA template... to introduce precise modifications by forming a complex within a cell and extending the DNA sequence with a template
Data Source
AI summary
The present invention relates to the field of genetic engineering. Specifically, the present invention relates to a genome editing system and method based on DNA polymerase. More specifically, the present invention relates to a method for site-directed introduction of a target modification into a genome by combining a DNA polymerase with a sequence-specific nuclease, and at the same time providing a DNA template sequence carrying the desired modification.