Cas13 RNA Editing With Adenosine Deaminase for Precise Targeting
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Solution Overview
Problem
Current genome-editing technologies lack affordability, ease of setup, scalability, and the ability to target multiple positions within the eukaryotic genome effectively, and existing RNA-targeting methods face challenges in specificity and delivery efficiency.
Innovation Solution
Development of an engineered RNA-targeting system using a catalytically inactive Cas13 protein fused with an adenosine deaminase to convert adenine to inosine in RNA sequences, utilizing guide molecules and direct repeat sequences for precise targeting and editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If DNA-targeting genome editing is used, then precise genome perturbations can be achieved, but the technology lacks affordability, ease of setup, and scalability
Solution Approach 1:
The invention extracts the essential function of genome editing by shifting from DNA-targeting to RNA-targeting. This extraction allows the system to achieve precise genetic element perturbation through RNA intermediates, simplifying the overall system while maintaining targeting precision. The RNA-targeting approach eliminates the need for complex DNA delivery and nuclear entry requirements.
Solution Approach 2:
The invention introduces RNA as an intermediary between the editing machinery and the genome. The RNA-targeting system uses guide RNA molecules to direct the editing complex to specific transcripts, serving as a mediator that simplifies delivery requirements and improves scalability compared to direct DNA-targeting methods.
2Adaptability or versatility
If existing RNA-targeting methods are used, then gene expression can be modulated, but specificity and delivery efficiency are compromised
Solution Approach 1:
The invention segments the RNA-targeting system into distinct functional modules: a guide RNA component for specificity and an adenosine deaminase component for editing activity. This segmentation allows independent optimization of each component, improving overall specificity while maintaining versatility in gene expression modulation applications.
Solution Approach 2:
The invention applies local quality by engineering the adenosine deaminase with specific mutations (e.g., E488Q in human ADAR2) to enhance its catalytic activity and specificity for adenosine deamination. This localized optimization of the enzyme's active site improves reliability without compromising the overall adaptability of the RNA-targeting system.
3Productivity
If adenosine deaminase is used for RNA editing, then adenine can be converted to inosine, but the system must be engineered to achieve desired specificity and activity
Solution Approach 1:
The invention changes key parameters of the adenosine deaminase enzyme through site-directed mutagenesis. Specific amino acid substitutions (such as E488Q in human ADAR2 or corresponding residues in other ADAR orthologs) are introduced to optimize catalytic efficiency and substrate specificity. These parameter changes enhance productivity while the modular nature of the system keeps engineering requirements manageable.
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 system provides efficient and specific RNA editing with reduced off-target effects, enabling therapeutic applications such as correcting genetic mutations and modifying gene expression, and is suitable for in vivo and ex vivo use.
Implementation Method 1
an adenosine deaminase or catalytic domain thereof, wherein the adenosine deaminase is modified to convert activity to a cytidine deaminase
Data Source
AI summary
The invention provides for systems, methods, and compositions for targeting and editing nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a RNA-targeting Cas13 protein, at least one guide molecule, and at least one adenosine deaminase protein or catalytic domain thereof.