Non-Cleavage Base Editing via Deaminase-Linked Cas9 Complex
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Solution Overview
Problem
Conventional genome editing techniques require double-stranded DNA breaks, leading to cytotoxicity, chromosomal rearrangements, and low efficiency in gene therapy, especially in primate ova and unicellular microorganisms, due to their reliance on cleavage-based methods.
Innovation Solution
A method utilizing a complex of a nucleic acid sequence-recognizing module and a nucleic acid base converting enzyme, such as CRISPR-Cas system with inactivated cleavage ability, for base conversion without DNA cleavage, allowing targeted nucleotide modifications in specific DNA sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional genome editing techniques using double-stranded DNA breaks are employed, then DNA sequence modification can be achieved, but cytotoxicity and chromosomal rearrangements occur
Solution Approach 1:
The patent extracts and removes the DNA cleavage function from the CRISPR-Cas9 system by using mutated Cas9 proteins (D10A and H840A mutants) that cannot cleave DNA. This leaves only the sequence recognition and base conversion functions, eliminating the harmful double-stranded breaks while preserving the targeted modification capability
Solution Approach 2:
The patent replaces the mechanical DNA cleavage mechanism with a chemical base conversion mechanism. Instead of breaking phosphodiester bonds through nuclease activity, the system uses deaminase enzymes to chemically convert bases (cytosine to uracil, adenine to hypoxanthine), achieving sequence modification without physical DNA strand breakage
2Manufacturing precision
If cleavage-based genome editing methods are used, then target gene modification is possible, but reliability in gene therapy is impaired
Solution Approach 1:
The patent converts the previously harmful effect of DNA breaks into a beneficial non-cleavage mechanism. By exploiting the natural base pairing rules and cellular repair mechanisms, the system uses the absence of cleavage to ensure reliability while still achieving precise base conversion through deaminase activity followed by error-prone repair
Solution Approach 2:
The patent introduces deaminase enzymes as intermediary molecules that facilitate base conversion without requiring DNA cleavage. These enzymes act as mediators between the CRISPR-Cas9 targeting system and the final base modification, enabling reliable gene therapy through a safer chemical conversion pathway
3Productivity
If artificial nucleases with DNA cleavage ability are employed, then genome editing can be performed, but the number of surviving cells is extremely small
Solution Approach 1:
The patent removes the DNA cleavage capability from the system by using mutated Cas9 proteins that retain binding and recruitment functions but lack endonuclease activity. This extraction of the harmful cleavage function allows genome editing to proceed with high cell survival rates
Solution Approach 2:
The patent changes the fundamental parameter of DNA modification from physical bond breaking to chemical base conversion. This parameter change from cleavage-based to conversion-based editing dramatically increases cell survival while maintaining editing efficiency through the use of deaminase enzymes
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 enhances safety by avoiding double-stranded DNA breaks and foreign DNA insertion, enabling efficient and precise nucleotide modifications across various species, including eukaryotic and prokaryotic cells, with high mutation introduction efficiency and applicability to multiple genomic regions.
Implementation Method 1
a method of modifying a targeted site of a double stranded DNA, comprising 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 a nucleic acid base converting enzyme are linked, with said double stranded DNA, to convert one or more nucleotides in the targeted site to other one or more nucleotides
Data Source
AI summary
The 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 a nucleic acid base converting enzyme are linked, 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.