High Throughput RNA Editing Screening Using ADAR Enzymes
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Solution Overview
Problem
Current gene therapy approaches using programmable DNA nucleases face challenges such as low efficiency in homologous recombination and the risk of introducing off-target mutations, along with immunogenicity concerns due to non-human origins, necessitating the development of methods that directly target RNA and utilize native host machinery.
Innovation Solution
The method involves identifying a guide RNA suitable for editing a target RNA by contacting a self-annealing RNA structure with an RNA editing entity, such as ADAR, which forms a guide-target RNA scaffold with structural features like hairpin loops and bulges, allowing for specific editing and high-throughput screening to optimize guide RNA design.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If programmable DNA nucleases are used to repair mutations, then gene editing can be achieved, but the efficiency of homologous recombination is low and off-target mutations are introduced
Solution Approach 1:
The patent replaces the mechanical DNA nuclease system with a chemical RNA editing system. Specifically, it uses ADAR enzymes (adenosine deaminases acting on RNA) to chemically modify RNA bases directly, eliminating the need for DNA nucleases and homologous recombination. This substitution resolves the contradiction by achieving high editing precision through direct RNA base modification while avoiding the low efficiency and off-target effects associated with DNA nuclease-based approaches.
Solution Approach 2:
The patent introduces guide RNA molecules as intermediaries to direct the ADAR editing process. The guide RNA contains a guide sequence that hybridizes to the target RNA, positioning the ADAR enzyme at the correct location. This intermediary system enables precise RNA editing without requiring DNA nucleases, thereby improving both precision and eliminating the need for inefficient homologous recombination.
2Reliability
If programmable DNA nucleases of non-human origin are used, then gene editing can be performed, but in vivo immunogenicity is raised
Solution Approach 1:
The patent changes the fundamental parameters of the editing system by switching from foreign DNA nucleases to native human RNA editing machinery. It uses human ADAR enzymes that are endogenous to the host cell, thereby eliminating immunogenicity while maintaining editing capability. The system leverages the host's own molecular machinery rather than introducing foreign proteins, resolving the contradiction between editing capability and immunogenicity.
Solution Approach 2:
The patent employs the host cell's own RNA editing machinery (ADAR enzymes) to perform the editing function. By using self-service, the system avoids introducing foreign proteins that would trigger immune responses. The endogenous enzymes are recruited to edit target RNAs through guide RNA direction, achieving editing capability without immunogenicity.
3Object-affected harmful factors
If RNA editing is performed using native host machinery, then immunogenicity is reduced, but the complexity of identifying suitable guide RNAs increases
Solution Approach 1:
The patent uses computational models to create virtual copies and predictions of guide RNA performance before actual experimentation. It employs algorithms to predict which guide RNAs will be most effective based on sequence complementarity and structural features, allowing high-throughput screening of potential guides. This copying approach simplifies the identification process by using computational predictions to guide physical experiments, reducing the practical complexity despite the inherent complexity of RNA structure prediction.
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 the specificity and efficiency of RNA editing, reducing off-target effects and immunogenicity by leveraging native RNA editing machinery, thereby improving the precision and safety of gene editing therapies.
Implementation Method 1
when the candidate guide RNA hybridizes to the target RNA, a hairpin loop is formed
Implementation Method 2
the RNA editing entity is: (a) an adenosine deaminase acting on RNA (ADAR)
Data Source
AI summary
Provided herein is a high throughput screening method for identifying guide RNAs (gRNAs) useful for editing a target RNA, wherein the editing is mediated by an RNA editing entity (e.g., a native human adenosine deaminase enzyme for a human subject).