SaCas9 SluCas9 Precise Exon Excision DMD Gene Editing
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Solution Overview
Problem
Current gene editing strategies for treating Duchenne muscular dystrophy (DMD) primarily focus on cutting at multiple sites or introducing frame-shifting mutations, lacking effective alternatives for precise genetic correction.
Innovation Solution
The use of Staphylococcus aureus (SaCas9) and Staphylococcus lugdunensis (SluCas9) Cas proteins with specific guide RNAs to excise small portions of the DMD gene, encoded on nucleic acid molecules, for targeted genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-based genome editing is used to treat DMD by cutting at multiple sites to excise large portions of the dystrophin gene, then the treatment can address significant genetic defects, but the excision precision is reduced and larger genetic disruptions occur
Solution Approach 1:
The invention divides the genome editing process into multiple targeted cutting sites within specific exons (44, 50, and 53) of the dystrophin gene. By segmenting the excision into controlled portions rather than removing large continuous sections, the method achieves precise removal of only the necessary defective segments while preserving the overall gene structure and function.
Solution Approach 2:
The patent applies local quality by targeting specific local regions (exons 44, 50, and 53) of the dystrophin gene for excision. The guide RNAs are designed to bind to specific sequences within these exons, ensuring that only the locally defective portions are removed rather than large portions of the entire gene, thereby maintaining precision while minimizing genetic material loss.
2Adaptability or versatility
If CRISPR-based genome editing is used to treat DMD by introducing frame-shifting mutations, then the treatment can disrupt mutant dystrophin expression, but alternative effective gene editing strategies are limited
Solution Approach 1:
Instead of using the conventional approach of introducing frame-shifting mutations to disrupt mutant dystrophin expression, this invention inverts the strategy by using precise excision of specific exons to restore the reading frame. By removing the defective portions rather than adding disruptions, the method achieves both effectiveness and creates a new versatile approach to DMD treatment.
Solution Approach 2:
The patent changes the key parameter of gene editing from introducing insertions/deletions that cause frame-shifting to making precise excisions that restore the reading frame. This parameter change in the editing approach provides a new effective strategy that differs from conventional methods while maintaining treatment reliability.
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 more precise and effective gene editing by using SaCas9 and SluCas9 with guide RNAs to target specific sequences in the DMD gene, potentially leading to improved treatment outcomes for DMD by reducing muscle degeneration and associated cardiomyopathy.
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
When a guide RNA and a Cas9 are expressed, the guide RNA will bind to Cas9 and direct it to the sequence complementary to the guide sequence, where it will then initiate a double-stranded break (DSB)
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 exons 44, 50, and 53 of the DMD gene are encompassed.