Base-Excision Genome Editing Without DNA Breaks
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genome editing techniques that rely on double-stranded DNA breaks are associated with cytotoxicity, chromosomal rearrangements, and low efficiency in gene modification, particularly in primate ovum and unicellular microorganisms, and existing methods using DNA glycosylase have limitations in mutation induction efficiency and specificity.
Innovation Solution
A method involving a complex of a nucleic acid sequence-recognizing module and a DNA glycosylase with low reactivity to unrelaxed double helix structures, using CRISPR-Cas system or other modules like zinc finger and TAL effector, to induce mutations by base excision reactions without cleaving double-stranded DNA, leveraging enzymes with CDG or TDG activity and AP endonuclease to enhance mutation frequency and reduce cytotoxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If double-stranded DNA breaks are used for genome editing, then gene modification can be achieved, but cytotoxicity and chromosomal rearrangements occur
Solution Approach 1:
The invention extracts and removes the DNA cleavage function from the nuclease enzyme, retaining only the sequence recognition capability. By using a mutated nuclease that cannot cleave DNA, the harmful double-stranded breaks are eliminated while the targeting function remains intact for guiding the base modification enzyme to the correct genomic location.
Solution Approach 2:
The invention introduces an intermediary enzyme (base modification enzyme such as cytidine deaminase or adenine deaminase) that performs the actual gene modification without causing DNA breaks. This intermediary enzyme is guided to the target site by the sequence-specific recognition module, enabling precise base conversion without the harmful effects of nuclease cleavage.
2Manufacturing precision
If DNA glycosylase is used for base excision, then mutation induction can occur, but the efficiency is low and specificity is limited
Solution Approach 1:
The invention merges the sequence-specific recognition capability of mutated nucleases with the base modification activity of deaminase enzymes. This combined system achieves both precise targeting and efficient mutation induction in a single integrated molecular complex, eliminating the need for separate guidance and modification components.
Solution Approach 2:
The invention changes the substrate specificity parameters of the enzyme system by using deaminase enzymes that can modify specific bases (cytosine or adenine) at the target site. This parameter change enables the system to induce transitions to uracil or hypoxanthine respectively, creating programmable mutation outcomes based on the chosen enzyme's specificity.
3Manufacturing precision
If foreign DNA fragments are inserted for genome modification, then gene function can be changed, but the process becomes complex and less safe
Solution Approach 1:
The invention extracts and eliminates the need for foreign DNA fragment insertion by using endogenous base modification enzymes. The modification is achieved through in situ base conversion rather than introducing exogenous genetic material, thereby simplifying the process and improving safety by avoiding foreign DNA integration.
Solution Approach 2:
The invention enables the cell's own DNA repair and modification systems to perform the gene function change. By inducing base modifications that create stop codons or alter amino acid sequences, the cell's natural transcription and translation machinery automatically processes the modification, eliminating the need for complex foreign DNA insertion and integration steps.
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 enables safe and efficient genome editing with reduced cytotoxicity and improved mutation induction, allowing for targeted modifications across several hundred bases without the need for foreign DNA insertion or double-stranded breaks, applicable to various cell types including mammalian cells.
Implementation Method 1
utilizing a base excision reaction
Data Source
AI summary
The present invention provides a method of modifying a targeted site of a double stranded DNA, including a step of contacting a complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a selected double stranded DNA and DNA glycosylase with sufficiently low reactivity with a DNA having an unrelaxed double helix structure (unrelaxed DNA) are bonded, with the double stranded DNA, to convert one or more nucleotides in the targeted site to other one or more nucleotides or delete one or more nucleotides, or insert one or more nucleotides into the targeted site, without cleaving at least one strand of the double stranded DNA in the targeted site.