Guide RNA Recruitment of Endogenous ADAR for Precise RNA Editing
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Solution Overview
Problem
Current genome and RNA editing technologies rely on exogenous proteins, which can pose risks due to aberrant effector activity, delivery limitations, and immunogenicity, and often result in low editing efficiency, requiring complicated chemical modifications.
Innovation Solution
A programmable approach using engineered RNA (dRNA) that is partially complementary to target transcripts to recruit endogenous ADAR proteins (ADAR1 or ADAR2) for targeted RNA editing, without introducing exogenous proteins, and includes specific chemical modifications to enhance efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If exogenous proteins are introduced for genome editing, then precise DNA cleavage and editing can be achieved, but delivery limitations and immunogenicity risks increase
Solution Approach 1:
The invention leverages the cell's own endogenous ADAR enzymes to perform RNA editing, eliminating the need to introduce exogenous deaminase proteins. The system uses engineered dRNA molecules that recruit endogenous ADAR1 or ADAR2 to specific target sites, allowing the cell to edit its own RNA without external protein intervention, thus avoiding immunogenicity while maintaining editing precision
Solution Approach 2:
The engineered dRNA acts as an intermediary molecule that bridges the gap between sequence-specific targeting and enzymatic editing. The dRNA contains a guide sequence complementary to the target RNA and recruits endogenous ADAR enzymes to the target site, enabling precise editing without requiring delivery of complex protein complexes
2Manufacturing precision
If conventional RNA editing methods are used, then A to G base editing can be achieved, but editing efficiency remains low and complicated chemical modifications are required
Solution Approach 1:
The invention optimizes key parameters of the editing system: (1) dRNA length (60-200 nucleotides) to balance stability and accessibility, (2) position of mismatched base pair (7-50 nucleotides from 3' end) to maximize ADAR recruitment efficiency, (3) degree of complementarity (80-100%) to ensure specific binding. These parameter optimizations enable high editing efficiency without requiring chemical modifications
Solution Approach 2:
The dRNA is designed with partial complementarity (80-100%) rather than perfect matching, creating a controlled mismatch that recruits ADAR enzymes. The mismatched base pair position is strategically placed 7-50 nucleotides from the 3' end to provide sufficient ADAR recruitment while maintaining specific binding, achieving high efficiency without excessive chemical stabilization
3Ease of operation
If short RNA sequences are used for editing, then delivery is simplified, but editing efficiency decreases
Solution Approach 1:
The invention identifies an optimal dRNA length parameter of 60-200 nucleotides that balances delivery simplicity with editing efficiency. This length is sufficient to provide stable binding and effective ADAR recruitment while remaining small enough for efficient cellular delivery, eliminating the need for overly complex delivery systems
4Measurement precision
If highly complementary RNA sequences are used, then binding specificity increases, but off-target effects increase
Solution Approach 1:
The dRNA is designed with heterogeneous properties: a highly complementary region (80-100% identity) for specific target binding, combined with a strategically positioned mismatched base pair 7-50 nucleotides from the 3' end. This local variation in complementarity creates a 'sweet spot' that enhances on-target ADAR recruitment while the controlled mismatch prevents excessive stability that would lead to off-target binding
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
Achieves efficient and precise RNA editing with minimal off-target effects, enabling point mutations, misfolding, or splicing changes in target RNAs, and functional protein production, while avoiding the risks associated with exogenous protein introduction.
Implementation Method 1
the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA
Implementation Method 2
ADAR removes the -NH2 group from an adenosine (A), converting A to inosine (I)
Data Source
Figure 1A~1D
Figure 1E
Figure 1F~1H
AI summary
Provided are methods for editing RNA by introducing a deaminase-recruiting RNA in a host cell for deamination of an adenosine in a target RNA. Further provided are deaminase-recruiting RNAs used in the RNA editing methods and compositions comprising the same.