Base Editing Complex for Precise DNA Modification Without Cleavage
Solution Overview
Problem
Current DNA modification techniques using CRISPR-Cas systems involve DNA cleavage, leading to cytotoxicity, unintended genome modifications, and limited efficiency in homologous recombination, restricting the type and site of mutations that can be introduced.
Innovation Solution
A method utilizing a complex of a nucleic acid sequence-recognizing module and a nucleic acid base converting enzyme or DNA glycosylase, which contacts a donor DNA with a target DNA to induce homologous recombination without cleaving the DNA strands, allowing for precise modification of targeted sites with high efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas systems are used for DNA modification, then targeted DNA cleavage and mutation introduction are achieved, but cytotoxicity increases and reliability decreases due to unintended genome modifications
Solution Approach 1:
The invention extracts and removes the DNA cleavage function from the CRISPR-Cas system by using dCas9 (dead Cas9) with inactivated nuclease activity. This separates the DNA binding function (guide RNA-directed targeting) from the DNA cutting function, allowing targeted modification without double-strand breaks, thereby reducing cytotoxicity and unintended modifications while maintaining targeting precision
Solution Approach 2:
The invention introduces an intermediary mechanism - using a deaminase enzyme as the actual modifying agent rather than direct Cas9 cleavage. The deaminase is recruited to the target site by the dCas9-guide RNA complex, where it catalyzes base conversion (e.g., C to U). This intermediary enzymatic process enables precise mutation introduction without the harmful effects of DNA cleavage
2Productivity
If Cas9 nuclease is used for genome editing, then efficient DNA cleavage occurs, but homologous recombination efficiency remains limited and cell survival decreases
Solution Approach 1:
The invention converts the harmful effect of DNA cleavage into a beneficial outcome by using the cell's natural DNA repair mechanisms. Instead of relying on error-prone NHEJ or low-efficiency HDR after DSB, the system uses deamination to create uracil lesions that trigger base excision repair (BER) and homologous recombination through a different pathway, achieving high-efficiency mutation introduction with improved cell survival
3Reliability
If deaminase-mediated base editing is used, then cytotoxicity is reduced, but the type and site of mutations are restricted
Solution Approach 1:
The invention enhances versatility by combining dCas9 (which can target any DNA sequence with appropriate guide RNA) with deaminase activity. This universal platform can be programmed to target different genomic locations and introduce various types of mutations (C to T, G to A transitions, and with engineered variants, other base conversions) by simply changing the guide RNA sequence, making the system adaptable to diverse editing needs
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 efficient and precise modification of targeted DNA sites with reduced cytotoxicity and increased accuracy, allowing for the introduction of various mutations and gene segments without the limitations of traditional methods.
Implementation Method 1
a nucleic acid base converting enzyme or DNA glycosylase, which contacts a donor DNA with a target DNA
Implementation Method 2
modification of a targeted site in a double-stranded DNA, which a cell has, using homologous recombination
Data Source
AI summary
The present invention provides a method for modifying a targeted site of a double-stranded DNA in a cell, the method including a step of bringing a complex in which a nucleic acid sequence-recognizing module that specifically binds to a selected target nucleotide sequence in a double-stranded DNA and a nucleic acid base converting enzyme or DNA glycosylase are linked, and a donor DNA containing an insertion sequence into contact with said double-stranded DNA, to substitute the targeted site with the insertion sequence, or insert the insertion sequence into said targeted site, without cleaving at least one strand of said double-stranded DNA in the targeted site.