Guide RNA for Mutant GUCY2D Allele Discrimination
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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 causing genetic disorders, such as cone-rod dystrophy, while preserving the functional allele.
Innovation Solution
Utilizing a method that employs naturally occurring nucleotide differences, specifically single nucleotide polymorphisms (SNPs), to differentiate between mutated and functional alleles, and using a CRISPR nuclease guided by an RNA molecule with a guide sequence portion to knock out the expression of the mutated allele.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used to target gene expression, then general gene knockdown is achieved, but the functional allele cannot be distinguished from the mutated allele
Solution Approach 1:
The guide RNA is designed with a guide sequence that specifically targets only the mutated allele by incorporating nucleotide differences unique to the mutation. This local differentiation in the guide sequence enables selective binding to the mutated allele while preserving the functional allele, achieving allele-specific knockdown without requiring complex multi-component systems
Solution Approach 2:
The guide RNA molecule is segmented into distinct functional regions: a guide sequence portion (17-20 nucleotides) that specifically recognizes the mutated allele, and a scaffold portion that binds the CRISPR nuclease. This segmentation allows the mutated allele to be specifically targeted while leaving the functional allele untouched, resolving the contradiction between precision and complexity
2Reliability
If CRISPR nuclease is used with standard guide RNA, then general DNA cleavage occurs, but specific inactivation of the mutated allele is not achieved
Solution Approach 1:
The guide RNA incorporates a guide sequence with 17-20 nucleotides that is specifically designed to match the mutated allele sequence while differing from the functional allele by at least one nucleotide. This local sequence differentiation ensures that the CRISPR nuclease-gRNA complex binds only to the mutated allele, achieving reliable specific inactivation without requiring overly complex guide RNA structures
Solution Approach 2:
The guide sequence is designed to be slightly longer than the minimum required for binding (17-20 nucleotides instead of the typical 20), providing enhanced specificity through additional nucleotide contacts. This partial extension of the binding region increases reliability of mutated allele targeting while maintaining manageable guide RNA 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 allows for the specific inactivation of the mutated allele, thereby reducing or preventing the disease phenotype associated with dominant genetic disorders, while maintaining the function of the healthy allele.
Implementation Method 1
a CRISPR nuclease guided by an RNA molecule with a guide sequence portion to knock out the expression of the mutated allele
Implementation Method 2
naturally occurring nucleotide differences, specifically single nucleotide polymorphisms (SNPs), to differentiate between mutated and functional alleles
Data Source
AI summary
Methods for inactivating a mutant human guanylate cyclase 2D (GUCY2D) allele comprising delivering a gRNA having a crRNA comprising at least 17 contiguous nucleotides set forth in any one of SEQ ID NOs: 237, 238, 241, 242, 247, 248, 394, 307, 413, 414, 417, 418, or 3011, compositions thereof, and methods of preventing, treating, ameliorating or slowing the progression of cone-rod dystrophies.