Allele-Specific LRRK2 Knockout via CRISPR Guide RNA
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Solution Overview
Problem
Current methods fail to effectively distinguish and knockout the dominant-mutated LRRK2 allele associated with monogenic Parkinson's disease, leading to inadequate treatment of the condition.
Innovation Solution
A method involving the use of CRISPR nuclease and a guide RNA molecule targeting specific nucleotide sequences in the LRRK2 gene to introduce double-strand breaks, allowing for the differentiation and inactivation of the mutated allele, thereby allowing expression of the functional protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional gene knockout methods are used, then the LRRK2 gene is targeted, but the mutated allele cannot be distinguished from the functional allele, leading to non-specific knockout
Solution Approach 1:
The guide RNA is designed to recognize specific local sequence variations (SNPs) present only in the mutated LRRK2 allele. This local sequence differentiation allows the CRISPR system to selectively target and knockout only the disease-causing allele while preserving the functional allele, achieving allele-specific discrimination without requiring complex additional components
Solution Approach 2:
The LRRK2 gene target site is segmented into specific nucleotide sequences, with the guide RNA designed to bind only to the unique sequence pattern present in the mutated allele. This segmentation of the target recognition process enables precise discrimination between mutated and functional alleles based on their sequence differences
2Reliability
If the CRISPR system is designed to target the mutated allele specifically, then allele-specific knockout is achieved, but the complexity of designing and delivering the system increases
Solution Approach 1:
The guide RNA molecule is designed to contain within its own sequence the information necessary to distinguish the mutated allele from the functional allele. The guide RNA's nucleotide sequence directly complements the mutated allele's sequence, allowing the system to self-identify and target the correct allele without requiring external guidance or complex regulatory components
Solution Approach 2:
The guide RNA acts as an intermediary between the CRISPR nuclease and the LRRK2 gene, translating the sequence differences between alleles into specific binding affinity. The guide RNA mediates the interaction by forming complementary base pairs only with the mutated allele's sequence, thereby directing the nuclease activity specifically to the disease-causing 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 enables the specific targeting and inactivation of the mutant LRRK2 allele, potentially treating monogenic Parkinson's disease by reducing the expression of the disease-causing protein and allowing the functional protein to be expressed.
Implementation Method 1
a CRISPR nuclease or a sequence encoding the CRISPR nuclease; and a first RNA molecule comprising a guide sequence portion having 17-50 nucleotides... wherein a complex of the CRISPR nuclease and the first RNA molecule affects a double strand break in the mutant allele of the LRRK2 gene
Data Source
AI summary
RNA molecules comprising a guide sequence portion having 17-50 contiguous nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-103,606 and compositions, methods, and uses thereof.