FGA Allele-Specific CRISPR Knockout for Amyloid Prevention
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Solution Overview
Problem
Dominant genetic disorders caused by mutations in the FGA gene, such as hereditary renal amyloidosis, are challenging to treat due to the requirement of knocking out a single abnormal allele while preserving the functional allele.
Innovation Solution
Utilizing CRISPR nuclease guided by an RNA molecule with a 17-20 nucleotide guide sequence to target and knockout the mutated FGA allele, allowing expression of the functional protein by introducing frameshift mutations or exon removal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional methods are used to knock out a single abnormal allele while preserving the functional allele, then the treatment of dominant genetic disorders becomes feasible, but the precision and specificity required to differentiate between mutated and functional alleles creates significant technical challenges
Solution Approach 1:
The guide RNA is designed to target a specific local region (splice site) of the FGA gene that is unique to the mutated allele. By focusing the CRISPR-Cas9 system on this particular location rather than the entire gene, the method achieves high specificity for the mutated allele while preserving the functional allele, thus resolving the contradiction between precision and complexity
Solution Approach 2:
The invention uses a guide RNA molecule that copies or mimics the natural RNA splicing signals (splice sites) found in the FGA gene. This guide RNA serves as a template that directs the Cas9 enzyme to the exact location needing correction, enabling precise allele differentiation without requiring complex diagnostic procedures
2Productivity
If CRISPR-Cas9 is used to target the mutated FGA allele, then knockout efficiency is improved, but the risk of off-target effects and unintended mutations increases
Solution Approach 1:
The guide RNA targets a very specific local region (the splice site sequence) of the FGA gene. This localized targeting approach concentrates the editing activity precisely where needed, maximizing knockout efficiency for the mutated allele while minimizing exposure of other genomic regions to potential off-target effects, thus maintaining genomic stability
Solution Approach 2:
The invention replaces traditional mechanical or chemical mutagenesis methods with the CRISPR-Cas9 molecular biology system. This substitution enables programmable, sequence-specific targeting through the guide RNA, allowing high-efficiency knockout of the mutated allele with reduced off-target effects compared to conventional approaches
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
Effectively inactivates the mutated FGA allele, preventing amyloid formation and reducing disease symptoms by producing a functional fibrinogen alpha subunit without aggregates, thereby treating AFib amyloidosis.
Implementation Method 1
CRISPR nucleases and RNA molecules, and methods for using the same to treat a subject with a genetic disorder
Implementation Method 2
an RNA molecule with a 17-20 nucleotide guide sequence to target and knockout the mutated FGA 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: 1-1990 and compositions, methods, and uses thereof.