Mutant Cas-PmCDA1 Complex for Base Editing Without DNA Breaks
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Solution Overview
Problem
Conventional genome editing methods for gram-positive bacteria often result in double-stranded DNA breaks, leading to cytotoxicity, chromosomal rearrangements, and high costs due to the need for foreign DNA insertion and deletion, which complicates genetic modification and increases production costs.
Innovation Solution
A method using a CRISPR-Cas system with a mutant Cas protein that lacks DNA cleavage ability, combined with Petromyzon marinus cytidine deaminase 1 (PmCDA1) for base conversion, allowing targeted nucleotide modifications without DNA breaks or foreign DNA insertion, enabling specific base conversion in gram-positive bacteria like Clostridium, Brevibacillus, and Corynebacterium.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional genome editing methods using artificial nucleases are used, then targeted gene modification can be achieved, but double-stranded DNA breaks occur causing cytotoxicity and chromosomal rearrangements
Solution Approach 1:
The patent extracts and removes the harmful DNA cleavage function from the Cas protein by using mutant Cas variants (Cas9 D10A/H840A, Cas12a R259A/R672A, Cas12b R320A/R783A) where specific amino acid residues are mutated to inactivate both DNA cleavage activities, while preserving the sequence recognition capability through guide RNA binding
Solution Approach 2:
The patent introduces an intermediary enzyme (cytidine deaminase APOBEC1 or PmCDA1) that performs the actual base conversion function. The mutant Cas protein serves as a positioning intermediary that guides the deaminase to the target site without causing DNA breaks, enabling precise base conversion through the intermediary enzyme rather than direct nuclease action
2Manufacturing precision
If foreign DNA insertion or deletion methods are used for genetic modification, then gene recombination can be achieved, but facility costs and waste disposal costs increase significantly
Solution Approach 1:
The patent extracts and eliminates the need for foreign DNA insertion and large-scale DNA deletion operations by using direct base conversion. The invention achieves gene modification through in-situ base substitution (C-to-T or G-to-A conversion) rather than inserting foreign DNA fragments or creating large deletions that would require costly facility infrastructure and waste disposal systems
Solution Approach 2:
The patent uses transient expression of the base conversion system components (mutant Cas protein and deaminase) delivered via plasmids or mRNA, which are temporary and do not require permanent foreign DNA integration. This approach avoids the need for expensive GMP-grade facility infrastructure and complex waste disposal systems required for stable foreign DNA integration methods
3Ease of manufacture
If DNA cleavage is used to promote homologous recombination, then foreign gene insertion becomes easier, but genome stability is compromised
Solution Approach 1:
The patent inverts the conventional approach by not using DNA cleavage to promote recombination, but rather using direct base conversion that does not require DNA breaks at all. This inverse approach achieves gene modification (e.g., creating stop codons, amino acid substitutions) directly through base substitution without compromising genome stability through cleavage-induced rearrangements
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 reduces toxicity and costs by avoiding DNA breaks, facilitating safer and more efficient genetic modifications in gram-positive bacteria, thereby lowering facility and waste disposal costs in industrial fermentations.
Implementation Method 1
a nucleic acid sequence-recognizing module, which is a CRISPR-Cas system comprising a mutant Cas in which both DNA cleavage abilities of Cas are inactivated
Implementation Method 2
a nucleic acid base converting enzyme, which is Petromyzon marinus cytidine deaminase 1 (PmCDA1)
Data Source
Figure 1
AI summary
The present invention provide a method of modifying a targeted site of gram-positive bacterium 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 given double stranded DNA and a nucleic acid base converting enzyme are bonded, with said 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 said targeted site, without cleaving at least one strand of said double stranded DNA in the targeted site, wherein the double stranded DNA is contacted with the complex by introducing the nucleic acid encoding the complex into the gram-positive bacterium. The present invention also provide a nucleic acid-modifying enzyme complex of a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a double stranded DNA of a gram-positive bacterium and a nucleic acid base converting enzyme bonded to each other, which complex is used for the method.