Dual Vector CRISPR-Cas System with Auto-Limiting Endonuclease
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Solution Overview
Problem
Current CRISPR-Cas systems face limitations in controlling off-target effects, complexity, and efficacy, particularly in therapeutic applications for monogenic disorders affecting non-replicative cells and tissues with limited regeneration capacity, such as those in the CNS.
Innovation Solution
A dual vector CRISPR-Cas system with a simple design using a first viral expression vector encoding a guide RNA and a second vector encoding an endonuclease enzyme, where the endonuclease expression is auto-limiting through self-cleaving target sequences, minimizing off-target effects and enabling precise gene editing in non-dividing cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR-Cas systems are used for gene editing in non-replicative cells, then gene editing capability is achieved, but off-target effects increase and editing precision decreases
Solution Approach 1:
The system divides the CRISPR-Cas components into separate expression vectors, with guide RNA encoded in one vector and endonuclease in another. This segmentation allows independent optimization of each component's expression timing and level, reducing off-target effects while maintaining editing capability in non-replicative cells
Solution Approach 2:
The system uses self-cleaving target sequences that automatically limit endonuclease expression after initial activation. This preliminary action establishes a self-regulating mechanism that prevents prolonged endonuclease activity, thereby reducing off-target effects before they can accumulate in non-replicative cells
2Productivity
If complex CRISPR-Cas systems are designed to improve editing efficacy, then editing efficiency increases, but system complexity increases
Solution Approach 1:
The system employs self-cleaving target sequences that automatically regulate endonuclease expression without requiring external control mechanisms. This self-service approach maintains high editing efficiency through autonomous feedback control while minimizing system complexity by eliminating the need for additional regulatory components
Solution Approach 2:
The dual vector system design is universally applicable to different CRISPR-Cas variants and target sequences. The same basic architecture of separate guide RNA and endonuclease vectors with self-cleaving regulation can be used across different disease models and cell types, achieving high editing efficiency without redesigning the entire system
3Duration of action of stationary object
If endonuclease expression is sustained to improve editing efficacy, then editing completeness increases, but off-target effects increase
Solution Approach 1:
The system creates a periodic expression pattern where endonuclease is initially expressed at high levels to achieve complete editing, then automatically downregulated through self-cleaving of the target sequence. This periodic action ensures sufficient duration for editing completion while limiting prolonged exposure that would cause off-target effects
Solution Approach 2:
The self-cleaving target sequence acts as a feedback mechanism that monitors endonuclease activity and automatically reduces expression once initial editing is achieved. This feedback control maintains appropriate endonuclease levels throughout the process, ensuring editing completeness while preventing excessive expression that would lead to off-target effects
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 accurate and efficient gene editing with reduced off-target effects, allowing for therapeutic correction of mutations in difficult-to-reach tissues and organs, including the CNS, thereby providing a promising approach for treating monogenic diseases like Alport syndrome, Pompe disease, Rett syndrome, and Parkinson's disease.
Implementation Method 1
a guide RNA (gRNA), which comprises a scaffold nucleotide sequence capable of binding an endonuclease enzyme, and a guide nucleotide sequence capable of hybridizing to a mutant genomic target sequence
Implementation Method 2
The gRNA directs an endonuclease enzyme, such as Cas9, towards the target sequence, resulting in cleavage of the mutant genomic target sequence
Implementation Method 3
The double-strand break is repaired by using as template the donor nucleotide sequence consisting of the wild-type sequence of the mutant genomic target sequence in order to replace at least one of the mutations in the target sequence
Data Source
AI summary
There is described a genome editing system, more particularly a dual vector CRISPR-Cas system suitable for gene editing-mediated correction of a mutant genomic target sequence in a target cell. There are further described a set of two viral particles and a host eukaryotic cell comprising the CRISPR-Cas system of the invention as well as the therapeutic use of this system, particularly for the treatment of genetic diseases.