Modified Guide RNAs for CRISPR Editing Stability
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Solution Overview
Problem
Existing guide RNAs for CRISPR genome editing face limitations such as rapid degradation, reduced stability, and increased immunogenicity, which hinder their therapeutic applications by affecting editing efficiency and inducing immune responses.
Innovation Solution
Development of chemically modified guide RNAs with extended nucleic acid (exNA) intersubunit linkages and extensive nucleotide modifications, including ribose, phosphate, and nucleobase modifications, to enhance stability, potency, and reduce off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If chemically modified guide RNAs are used, then stability and genome editing efficacy are improved, but immunogenicity may increase
Solution Approach 1:
The patent applies parameter changes by systematically modifying the chemical structure of guide RNA molecules through various modifications including 2'-O-methyl, 2'-fluoro, phosphorothioate, and exNA linkages at different positions and densities. These parameter changes in chemical composition and structure enable optimization of both stability and immunogenicity profiles while maintaining genome editing efficacy.
Solution Approach 2:
The patent employs composite materials by creating guide RNAs with heterogeneous chemical modifications combining multiple types of modifications (ribose modifications, phosphate modifications, nucleobase modifications, and intersubunit linkages) within a single molecule. This composite approach allows simultaneous achievement of enhanced stability, reduced immunogenicity, and maintained editing activity.
2Stability of the object's composition
If guide RNAs are heavily chemically modified, then stability is improved, but editing efficiency may be reduced
Solution Approach 1:
The patent applies local quality by implementing non-uniform chemical modification patterns where different regions of the guide RNA molecule receive different types and densities of modifications. For example, the seed region may have minimal modifications to preserve binding efficiency, while other regions receive heavier modifications for stability, allowing simultaneous optimization of both properties.
Solution Approach 2:
The patent systematically varies chemical modification parameters including type, position, and density to find optimal balances between stability and editing efficiency. This includes testing different modification patterns such as 2'-O-methyl at specific positions, phosphorothioate linkages at defined locations, and exNA intersubunit linkages to achieve desired stability without compromising activity.
3Productivity
If existing guide RNAs are used, then genome editing activity is maintained, but rapid degradation occurs in circulation and cells
Solution Approach 1:
The patent applies preliminary action by pre-modifying guide RNAs with stabilizing chemical groups before administration. These modifications including 2'-O-methyl, 2'-fluoro, phosphorothioate, and exNA linkages are incorporated during synthesis to preemptively protect against nucleolytic degradation that would otherwise occur rapidly in circulation and cellular environments, thereby extending half-life while maintaining activity.
Solution Approach 2:
The patent uses composite materials by combining multiple types of chemical modifications in guide RNA molecules to create a multi-layered protection system against degradation. This composite modification strategy addresses various degradation pathways simultaneously, providing comprehensive stability enhancement while preserving genome editing functionality.
Data Source
AI summary
Extended Nucleic Acid (exNA)-modified crRNAs and tracrRNAs are provided. Methods of using the crRNAs and tracrRNAs for genome editing with a CRISPR nuclease and kits for performing the same are also provided.