CRISPR-Cas9 Guide RNA for FXN Gene Editing
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Solution Overview
Problem
Current genome engineering techniques for addressing Frataxin (FXN) related disorders lack safety and efficacy, particularly in achieving permanent corrections with minimal treatment interventions.
Innovation Solution
The use of single-molecule guide RNAs and CRISPR/Cas9 endonucleases to introduce specific single-strand or double-strand breaks in the FXN gene, enabling permanent deletions, insertions, or mutations to correct or eliminate aberrant FXN gene products, thereby treating conditions like Friedreich's Ataxia.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If random insertion technologies are used to introduce transgenes, then the simplicity of the method is maintained, but the precision and safety of gene modification deteriorates due to disruption of normal gene regulation
Solution Approach 1:
The patent introduces guide RNA as an intermediary molecule that mediates between the Cas9 endonuclease and the target FXN gene. The guide RNA contains a spacer sequence complementary to the target site, directing Cas9 to the precise location requiring modification while preventing random genomic insertions and their harmful effects
2Manufacturing precision
If zinc finger nucleases or TALENs are used to achieve specific DNA modification, then the precision of gene targeting is improved, but the device complexity and difficulty of construction increases
Solution Approach 1:
The patent segments the gene targeting function into two independent components: the Cas9 endonuclease responsible for DNA cutting and the guide RNA responsible for target recognition. This segmentation allows the guide RNA to be easily redesigned by simply changing the spacer sequence, eliminating the complex protein engineering required for ZFNs and TALENs while maintaining high precision targeting
Solution Approach 2:
The Cas9 endonuclease serves as a universal tool that can target multiple different genes by simply changing the guide RNA spacer sequence. This multi-functionality eliminates the need to construct different complex nuclease proteins for each target site, significantly reducing device complexity while maintaining precision across multiple applications
3Reliability
If multiple treatment interventions are used to achieve permanent genome correction, then the reliability of correction is improved, but the duration of treatment and patient burden increases
Solution Approach 1:
The patent introduces all necessary components (Cas9 endonuclease and guide RNA with spacer sequence) in a single treatment intervention that performs the complete genome editing process in one administration. The Cas9-gRNA complex directly targets and modifies the FXN gene in a single action, eliminating the need for multiple sequential treatments while maintaining reliable permanent correction
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 allows for precise and permanent editing of the FXN gene, potentially curing FXN-related disorders with a single treatment by restoring wild-type FXN protein function or reducing aberrant gene expression.
Implementation Method 1
one or more S. pyogenes Cas9 endonuclease or one or more polynucleotide encoding the one or more S. pyogenes Cas9 endonuclease
Data Source
Figure 1A~1B
Figure 2A
Figure 2B
AI summary
The present application provides materials and methods for treating a patient with one or more condition associated with FXN whether ex vivo or in vivo. In addition, the present application provides materials and methods for editing and/or modulating the expression of FXN gene in a cell by genome editing.