VEGFA-Targeting Guide RNA for Editing Efficiency and Safety
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Solution Overview
Problem
Current CRISPR/Cas systems face challenges in achieving high editing efficiency and safety for disease therapies, particularly in targeting VEGFA to treat conditions like neovascular age-related macular degeneration.
Innovation Solution
A guide RNA (gRNA) with specific sequences, exhibiting at least 80% to 100% sequence identity to SEQ ID NOs: 10-230, is engineered to hybridize with VEGFA, targeting conserved and splicing sites in human and mouse VEGFA RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas systems are used for disease therapy, then gene editing capability is achieved, but editing efficiency and safety are insufficient
Solution Approach 1:
The patent optimizes gRNA sequence parameters including length (20-26 nucleotides), GC content (40-60%), and secondary structure characteristics to enhance both editing efficiency and safety. Specific sequence identity thresholds (80-100% to SEQ ID NOs: 10-230) are established to achieve reliable target recognition while maintaining high editing activity
Solution Approach 2:
The patent designs gRNA with differentiated regional properties: a target-specific guide sequence region for precise binding, a scaffold region for Cas protein interaction, and optimized structural elements. This local differentiation allows simultaneous optimization of specificity (safety) and binding affinity (efficiency) in different parts of the gRNA molecule
2Measurement precision
If gRNA sequences with high identity to VEGFA are designed, then target specificity is improved, but off-target effects may increase
Solution Approach 1:
The patent employs gRNA sequences with 80-100% identity to VEGFA target sites, using partial identity ranges rather than requiring perfect matches. This allows optimization where 90-100% identity provides the best balance between on-target specificity and off-target minimization, rather than universally requiring 100% identity
Solution Approach 2:
The patent utilizes bioinformatic algorithms to predict and evaluate potential off-target binding sites by comparing gRNA sequences against genomic databases. This feedback mechanism allows selection of gRNA variants that maintain high VEGFA specificity while minimizing off-target effects through computational screening before experimental validation
3Reliability
If intravitreal injection of VEGF antibody drugs is performed frequently, then treatment efficacy is maintained, but patient burden and treatment complexity increase
Solution Approach 1:
The patent employs gene editing to permanently modify VEGFA expression in retinal cells, achieving long-term therapeutic effect from a single or limited number of administrations. This preliminary genetic modification eliminates the need for repeated antibody injections, reducing treatment complexity while maintaining sustained efficacy
Solution Approach 2:
The patent uses CRISPR/Cas gene editing to create a permanent copy of the therapeutic effect at the genomic level, where the edited gene continues to produce the desired phenotype without requiring continuous external drug administration. This genetic copy replaces the need for repeated pharmacological interventions
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 gRNA enhances the editing efficiency and safety of CRISPR/Cas systems, providing a potential treatment for conditions like neovascular age-related macular degeneration by inhibiting VEGFA expression.
Implementation Method 1
the guide sequence is engineered to hybridize with a target RNA
Data Source
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Figure 4B~4C
AI summary
Disclosed in the present disclosure are a gRNA targeting VEGFA, a gene editing system, and the use thereof. The gRNA in the present disclosure comprises a guide sequence which has at least 80% sequence identity to any one of SEQ ID NOs: 10-18 and 40-230. The gene editing system in the present disclosure comprises the gRNA of the present disclosure and an encoding sequence thereof, and an RNA-guided nuclease and an encoding sequence thereof.