Guide RNA Targeting Mutant IMPDH1 Allele for Retinitis Pigmentosa
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Solution Overview
Problem
Current methods lack an effective approach to distinguish and knock out the expression of a dominant-mutated allele, which causes genetic disorders such as retinitis pigmentosa.
Innovation Solution
Utilizing a method that employs a guide RNA molecule with 17-20 nucleotides to specifically target a single nucleotide polymorphism (SNP) between a mutated allele and a functional allele, in conjunction with a CRISPR nuclease, to degrade the mRNA of the mutated allele.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used to target genetic alleles, then it is difficult to distinguish between mutated and functional alleles, but the patent employs a guide RNA molecule with 17-20 nucleotides that specifically targets a single nucleotide polymorphism (SNP) to achieve precise allele discrimination
Solution Approach 1:
The guide RNA is designed to target a specific local region (17-20 contiguous nucleotides) containing the SNP that distinguishes the mutated allele from the functional allele. This localized targeting approach enables precise allele discrimination without requiring complex systemic changes to the CRISPR system.
Solution Approach 2:
The patent specifies a particular parameter range for the guide RNA length (17-20 nucleotides) to optimize binding specificity to the SNP-containing sequence. This parameter optimization ensures high discrimination precision while maintaining manageable design complexity.
2Reliability
If the mutated allele is targeted for knockout, then expression of the mutated protein is eliminated, but there is a risk of affecting the functional allele; the patent uses allele-specific targeting to knock out only the mutated allele while preserving functional allele expression
Solution Approach 1:
The guide RNA targets a unique local sequence (17-20 nucleotides) that is present in the mutated allele but not in the functional allele, ensuring that the CRISPR nuclease activity is restricted to the mutated allele only. This local specificity prevents off-target effects on the functional allele.
Solution Approach 2:
The guide RNA acts as an intermediary that recognizes and binds specifically to the SNP-containing sequence of the mutated allele, directing the CRISPR nuclease activity exclusively to the mutated allele while leaving the functional allele unaffected.
3Productivity
If CRISPR nuclease is used to degrade mRNA, then expression of the targeted allele is knocked out, but the method requires precise delivery to specific cells; the patent employs specific delivery methods for retinal cells to treat retinitis pigmentosa
Solution Approach 1:
The patent extracts and degrades only the specific mRNA transcript from the mutated allele through CRISPR-mediated cleavage, leaving other cellular components and the functional allele's mRNA intact. This selective extraction approach achieves high degradation efficiency while minimizing operational complexity.
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 effectively distinguishes between mutated and functional alleles, knocks out the expression of the mutated allele, and allows for the expression of the functional allele, potentially treating or preventing dominant genetic disorders like retinitis pigmentosa.
Implementation Method 1
a guide RNA molecule with 17-20 nucleotides to specifically target a single nucleotide polymorphism (SNP) between a mutated allele and a functional allele
Implementation Method 2
in conjunction with a CRISPR nuclease, to degrade the mRNA of the mutated allele
Data Source
AI summary
RNA molecules comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and compositions, methods, and uses thereof.