Self-Inactivating CRISPR-Cas Vectors for Precise Repeat Editing
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Solution Overview
Problem
Current genome-editing technologies are not affordable, easy to set up, and not scalable for targeting multiple positions within the eukaryotic genome, limiting their effectiveness in addressing genetic disorders.
Innovation Solution
A self-inactivating CRISPR-Cas composition comprising engineered CRISPR-Cas systems with specific guide sequences and promoters, delivered via viral vectors, to target and edit nucleotide repeat disorders in a controlled manner, minimizing off-target activity and duration of expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If current genome-editing technologies (designer zinc fingers, TALEs) are used, then targeted genome perturbation is achieved, but affordability, ease of setup, and scalability are poor
Solution Approach 1:
The patent uses CRISPR guide RNA sequences as programmable copies of target DNA sequences, allowing the system to recognize and bind to specific genomic locations without requiring complex protein engineering. The guide RNA acts as a simple nucleic acid copy of the target site, making the system easier to design and implement while maintaining high targeting precision through complementary base pairing
Solution Approach 2:
The patent changes the fundamental parameter of genome editing from protein-based recognition (zinc fingers, TALEs) to nucleic acid-based recognition (CRISPR guide RNA). This parameter change simplifies the system design, as nucleic acid sequences can be easily synthesized and modified, improving ease of setup and scalability while maintaining targeting accuracy through sequence complementarity
2Productivity
If CRISPR-Cas system is delivered via viral vectors, then delivery efficiency is improved, but risk of insertional mutagenesis and immune response increases
Solution Approach 1:
The patent segments the CRISPR-Cas system into multiple separate viral vector components, each carrying different elements (Cas9 gene, guide RNA, self-inactivation sequence). This segmentation allows controlled expression and reduces the risk of insertional mutagenesis by distributing genetic elements across multiple vectors rather than delivering a large integrated construct
Solution Approach 2:
The patent introduces self-inactivating sequences as intermediary elements that control the expression and duration of CRISPR-Cas activity. These sequences act as mediators that automatically terminate Cas9 expression after a defined period, reducing long-term risks of insertional mutagenesis and immune response while maintaining high delivery efficiency through viral vectors
3Reliability
If self-inactivating sequences are included, then off-target activity is reduced, but system complexity increases
Solution Approach 1:
The patent implements self-inactivating sequences that enable the CRISPR-Cas system to automatically regulate its own expression and termination. The system serves itself by incorporating sequences that trigger Cas9 mRNA degradation or protein inactivation after a defined period, reducing off-target activity without requiring external control mechanisms, thus limiting the increase in system complexity
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 CRISPR-Cas system effectively edits nucleotide repeat disorders by reducing off-target effects and limiting expression duration, providing a scalable and efficient approach for treating a range of genetic conditions.
Implementation Method 1
the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence
Implementation Method 2
the tracr mate sequence that can be hybridized to the tracr sequence
Data Source
Figure 1
Figure 2A~2B
Figure 3A~3C
AI summary
The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a SIN CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing SIN CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.