Uracil Stabilizing Proteins in Fusion Systems
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Solution Overview
Problem
Current genome editing tools, such as the rAPOBEC1-Cas9 fusion system, face challenges in stabilizing uracil residues in DNA, leading to the removal of uracil by cellular Uracil DNA Glycosylase, which hampers the introduction of desired C>T mutations.
Innovation Solution
The use of Uracil Stabilizing Proteins (USPs) that inhibit Uracil DNA Glycosylase, allowing the uracil to remain present long enough for replication to introduce the desired C>T mutation, incorporated into fusion proteins with DNA-binding and deaminase polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If RNA-guided nucleases are used to introduce double-stranded breaks for genome editing, then targeted DNA editing capability is improved, but the complexity of generating chimeric nucleases for each target sequence increases
Solution Approach 1:
The patent uses guide RNA as an intermediary molecule that directs the Cas9 nuclease to specific target sequences. This separates the targeting function (handled by programmable guide RNA) from the cutting function (handled by the Cas9 enzyme), eliminating the need to generate new chimeric nucleases for each target and greatly simplifying the system's adaptability
Solution Approach 2:
The Cas9 nuclease serves as a universal platform that can target any DNA sequence by simply changing the guide RNA sequence. This multi-functional design allows a single enzyme to perform numerous different targeting tasks, resolving the contradiction between versatility and complexity
2Productivity
If cytosine deaminase is used to convert cytosine to uracil for C>T mutations, then the desired genetic modification is achieved, but cellular Uracil DNA Glycosylase removes the uracil residue, creating abasic sites and reducing mutation efficiency
Solution Approach 1:
The patent applies preliminary anti-action by using the uracil stabilizing protein to prevent the harmful action of Uracil DNA Glycosylase before it can remove the uracil residue. This protective measure ensures the uracil remains in place long enough for the desired C>T mutation to be fixed during DNA replication
Solution Approach 2:
The patent converts the potentially harmful presence of uracil (which could be recognized and removed by repair enzymes) into a beneficial intermediate state. By stabilizing the uracil residue, the system allows the uracil to serve as a stable intermediate that directs accurate C>T mutation incorporation during replication
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
The stabilization of uracil residues increases the efficiency of introducing C>T mutations by preventing the creation of abasic sites, thereby enhancing the accuracy and likelihood of the desired genetic modifications.
Implementation Method 1
a deaminase polypeptide, and a uracil stabilizing polypeptide
Implementation Method 2
Uracil stabilizing proteins and active fragments and variants thereof and methods of use
Data Source
AI summary
Compositions and methods comprising uracil stabilizing polypeptides for targeted editing of nucleic acids are provided. Compositions comprise uracil stabilizing polypeptides. Also provided are fusion proteins comprising i) a DNA-binding polypeptide; ii) a deaminase; and iii) a uracil stabilizing polypeptide (USP). The fusion proteins include RNA-guided nucleases fused to deaminases and further fused to a USP, optionally in complex with guide RNAs. Compositions also include nucleic acid molecules encoding the USPs or the fusion proteins. Vectors and host cells comprising the nucleic acid molecules encoding the USPs or the fusion proteins are also provided.