SaCas9 and SluCas9 Guide RNA Pairs for Exon 51 Excision
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Solution Overview
Problem
Current gene editing strategies for Duchenne muscular dystrophy (DMD) primarily focus on cutting large portions of the dystrophin gene or introducing frame-shifting mutations, lacking effective alternatives for treating the disease.
Innovation Solution
The use of Staphylococcus aureus (SaCas9) and Staphylococcus lugdunensis (SluCas9) Cas proteins with pairs of guide RNAs designed to excise small portions of the DMD gene, encoded on single or separate nucleic acid molecules, to potentially correct or knockout the mutated gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-based gene editing cuts at multiple different sites to excise large portions of the dystrophin gene, then the mutated gene can be knocked out or corrected, but the precision and control over the excision sites is reduced
Solution Approach 1:
The patent divides the dystrophin gene into multiple exons and uses multiple guide RNAs targeting specific sites within Exon 51. This segmentation allows precise control over which portions of the gene are excised while maintaining the ability to achieve functional correction through combination effects of multiple targeted cuts.
Solution Approach 2:
The patent employs different guide RNA sequences that are complementary to specific locations within Exon 51, creating locally optimized cutting sites. Each guide RNA is designed to target a specific sequence context, allowing precise local modification while maintaining overall gene structure integrity.
2Reliability
If CRISPR-based gene editing introduces frame-shifting mutations at a single site, then the dystrophin gene function can be disrupted, but the precision and controllability of the mutation outcome is reduced
Solution Approach 1:
The patent uses a dynamic approach where the outcome of the editing depends on the cell's repair mechanism choice. By providing multiple guide RNAs targeting Exon 51, the system allows cells to dynamically select different repair pathways (NHEJ or HDR), enabling control over the final mutation outcome based on cellular context and treatment conditions.
Solution Approach 2:
The patent changes the parameter of guide RNA sequence composition and targeting location to control the editing outcome. By selecting specific guide RNAs with particular sequences and positions, the system can influence whether frame-shifting mutations occur, whether specific splice sites are affected, and what the final functional outcome will be.
3Measurement precision
If multiple guide RNAs are used to target Exon 51, then precise excision of small portions can be achieved, but the complexity of the guide RNA system increases
Solution Approach 1:
The patent combines multiple guide RNAs into a single vector or nucleic acid molecule, allowing simultaneous delivery of multiple targeting sequences. This merging approach maintains the precision benefits of multiple targeted sites while reducing the operational complexity of delivering and managing separate guide RNA components.
Solution Approach 2:
The guide RNA system is designed with multi-functionality, where a single guide RNA can potentially serve multiple purposes depending on the cellular context and repair pathway utilized. The same guide RNA sequence can lead to different outcomes (frame-shifting mutations, splice site disruptions, or precise excisions) based on cellular conditions, reducing the need for entirely different editing components.
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 offers a new method for treating DMD by precisely targeting and editing the dystrophin gene, potentially reducing muscle degeneration and cardiomyopathy associated with the disease.
Implementation Method 1
The approximately 20 nucleotides at the 5′ end of the guide RNA serves as the guide or spacer sequence that can be any sequence complementary to one strand of a genomic target location
Implementation Method 2
CRISPR-based genome editing can provide sequence-specific cleavage of genomic DNA using a Cas9 and a guide RNA
Implementation Method 3
To repair these breaks, cells typically use an error prone mechanism of non-homologous end joining (NHEJ) which can lead to disruption of function in the target gene through insertions or deletion of codons
Data Source
AI summary
Compositions and methods for treating Duchenne Muscular Dystrophy (DMD) and excising small portions of exon 51 of the DMD gene are encompassed.