CRISPR Guide RNA for SAMD9 Allele Discrimination
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Solution Overview
Problem
Current methods fail to effectively distinguish and target dominant-mutated alleles causing genetic disorders like MIRAGE syndrome, leading to inadequate treatment options for such conditions.
Innovation Solution
A method utilizing CRISPR nuclease and a guide RNA molecule to introduce a double-strand break in the mutant allele of the SAMD9 gene, specifically targeting SNP positions to differentiate between functional and mutated alleles, thereby knocking out the expression of the mutated protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods are used to target genetic alleles, then treatment coverage is limited, but the ability to distinguish and target dominant-mutated alleles specifically is insufficient
Solution Approach 1:
The guide RNA is designed to recognize specific local nucleotide differences (SNPs) within the target allele, allowing precise discrimination between dominant-mutated and functional alleles by focusing on localized sequence variations rather than requiring comprehensive allele differentiation
Solution Approach 2:
The CRISPR system segments the targeting function into modular components: the Cas9 nuclease provides the cutting mechanism while the customizable guide RNA provides allele-specific recognition, allowing the system to be adapted to different alleles by simply changing the guide sequence rather than redesigning the entire system
2Manufacturing precision
If CRISPR nuclease is used to create double-strand breaks, then allele inactivation precision is improved, but the complexity of the system increases
Solution Approach 1:
The guide RNA acts as an intermediary that bridges the Cas9 nuclease and the target allele, providing sequence-specific recognition without requiring the Cas9 protein itself to be allele-specific. This separates the recognition function from the cutting function, simplifying the overall system design while maintaining high precision
Solution Approach 2:
The guide RNA sequence is designed to copy or match the specific nucleotide sequence of the target allele, allowing the system to recognize and target the correct allele through sequence complementarity rather than requiring complex structural recognition mechanisms
3Reliability
If dominant-mutated alleles are targeted for knockout, then disease treatment effectiveness is improved, but the risk of affecting functional alleles increases
Solution Approach 1:
The guide RNA targets specific local nucleotide differences (SNPs) that are unique to the dominant-mutated allele, creating a highly specific recognition pattern that minimizes the risk of off-target effects on functional alleles while maintaining effective disease treatment
Solution Approach 2:
The system exploits the asymmetric nucleotide differences between the dominant-mutated allele and the functional allele at specific SNP positions, designing guide RNAs that are asymmetric in their recognition requirements to ensure they only bind to the mutated allele and not the functional allele
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 inactivation of the mutant SAMD9 allele, potentially treating MIRAGE syndrome by enabling the expression of functional protein, thereby ameliorating or preventing the disease phenotype.
Implementation Method 1
a complex of the CRISPR nuclease and the first RNA molecule affects a double strand break in the mutant allele of the SAMD9 gene
Data Source
AI summary
RNA molecules comprising a guide sequence portion having 17-50 contiguous nucleotides containing nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-20640 and compositions, methods, and uses thereof.