Cas12f1 Dystrophin Exon 51 Editing for Single-Vector Delivery
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Solution Overview
Problem
Current CRISPR technologies face challenges in achieving high intracellular gene editing activity and efficient packaging of the gene editing system into a single vector for treating Duchenne muscular dystrophy, particularly in deleting exon 51 of the dystrophin gene, leading to temporary therapeutic effects and adverse side effects.
Innovation Solution
A CRISPR/Cas12f1 gene editing system utilizing a hypercompact nucleic acid cleavage protein, such as UnCas12f1 or CWCas12f1, combined with engineered guide RNAs, targets regions upstream and downstream of exon 51 in the dystrophin gene to enhance deletion efficiency, allowing in vivo delivery using a single vector like the adeno-associated virus (AAV) vector.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional CRISPR technologies are used for gene editing, then gene editing capability is achieved, but intracellular gene editing activity is low and the system cannot be efficiently packaged into a single vector
Solution Approach 1:
The patent segments the gene editing system into a compact Cas12f1 protein (9.5-11.5 kDa) and a minimized guide RNA (30-40 nucleotides), allowing both components to be packaged into a single AAV vector while maintaining high intracellular editing activity. This segmentation resolves the contradiction by dividing the system into smaller functional units that fit within vector constraints.
Solution Approach 2:
The patent extracts and utilizes only the essential functional components of the CRISPR system - the minimal Cas12f1 protein domain and the critical guide RNA sequence - removing non-essential elements. This extraction enables efficient single-vector packaging while preserving gene editing capability.
2Productivity
If RNA-based therapeutics are used for exon skipping, then temporary treatment effect is achieved, but the treatment duration is short and adverse effects occur
Solution Approach 1:
The patent replaces the RNA-based antisense oligonucleotide mechanism with a DNA-based CRISPR/Cas12f1 gene editing mechanism. This substitution transitions from temporary RNA-protein interaction to permanent DNA-level gene modification, achieving sustained therapeutic effects without the transient limitations of RNA therapeutics.
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
The system achieves efficient deletion of exon 51 in the dystrophin gene, enabling production of a functional protein, with improved nucleic acid cleavage efficiency and reduced vector size requirements, thus treating Duchenne muscular dystrophy effectively while minimizing adverse effects.
Implementation Method 1
an engineered guide RNA comprising a first guide sequence that hybridizes to a target sequence in a dystrophin gene
Implementation Method 2
an endonuclease comprising Cas12f1 or a variant protein thereof
Data Source
AI summary
A gene editing system for treating Duchenne muscular dystrophy, and a method for treating the disease using the gene editing system are disclosed. The system and method have the effects of making it possible to package the gene editing system in a single vector by editing the dystrophin gene using a CRISPR/Cas12f1 or TaRGET system, as well as making it possible to produce the dystrophin protein having a normal function by preventing the production of a stop codon of exon 51 through the skipping of exon 51, and thus can be useful for treating Duchenne muscular dystrophy.