CRISPR-Cas Delivery to Post-Mitotic Cells via Viral Vectors
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Solution Overview
Problem
Current genome-editing techniques, such as designer zinc fingers and TALEs, are complex and costly, lacking scalability and ease of use for targeting multiple positions within the eukaryotic genome, necessitating the development of more affordable and efficient genome engineering technologies.
Innovation Solution
The CRISPR-Cas system, which uses a single Cas enzyme programmed by a short RNA molecule to target specific DNA sequences, simplifies genome editing by enabling effective modification of polynucleotides in various cell types and tissues, including post-mitotic cells like those in the brain and kidney, through optimized guide RNAs and chimeric Cas9 enzymes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current genome-editing techniques (designer zinc fingers, TALEs) are used, then genome editing can be achieved, but the complexity and cost increase significantly
Solution Approach 1:
The patent uses a simplified CRISPR-Cas system that copies the essential function of complex genome-editing proteins through a single Cas enzyme guided by RNA molecules. This replaces the need for complex designer zinc fingers or TALEs with a more straightforward RNA-guided system, reducing overall system complexity while maintaining genome editing capability
Solution Approach 2:
The patent extracts and utilizes only the essential nuclease activity from the CRISPR system (Cas enzyme) separated from the complex protein-DNA recognition systems. By taking out just the core functional element (Cas enzyme guided by RNA) and removing unnecessary complexity, the system achieves genome editing with reduced complexity and cost
2Reliability
If current genome-editing techniques are used, then genome editing can be performed, but scalability and ease of use for targeting multiple positions deteriorate
Solution Approach 1:
The patent employs a universal CRISPR-Cas system where a single Cas enzyme can be programmed to target multiple different genomic positions by simply changing the guide RNA sequence. This multi-functional approach allows the same core enzyme to perform genome editing at various locations throughout the genome, significantly improving scalability compared to position-specific protein systems
Solution Approach 2:
The patent utilizes parameter changes in the guide RNA sequence to redirect the Cas enzyme to different target positions. By modifying the RNA guide sequence rather than creating entirely new protein systems, the same Cas enzyme can be rapidly reprogrammed for multiple targeting positions, enhancing productivity and ease of use
3Productivity
If CRISPR-Cas system is used for genome editing, then efficiency is improved, but toxicity and off-target modifications may increase
Solution Approach 1:
The patent optimizes the CRISPR-Cas system by making local improvements to the guide RNA design and Cas enzyme configuration. These localized optimizations enhance on-target specificity and reduce off-target effects by fine-tuning the interaction between the guide RNA and target DNA, thereby improving efficiency while minimizing harmful effects
Solution Approach 2:
The patent incorporates feedback mechanisms through optimized guide RNA design that enhances specificity. By designing guide RNAs with higher specificity and using controlled delivery methods, the system can monitor and reduce off-target effects, balancing efficiency improvement with reduced toxicity
4Device complexity
If CRISPR-Cas system is used, then simplicity and affordability are improved, but delivery to post-mitotic cells becomes challenging
Solution Approach 1:
The patent uses viral vectors as intermediary delivery vehicles to transport the CRISPR-Cas components into post-mitotic cells. These viral intermediaries overcome the delivery barrier by efficiently transducing non-dividing cells, enabling the simple and affordable CRISPR system to access target cells that are otherwise difficult to transfect
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 enhances the specificity and efficiency of genome editing, reducing toxicity and off-target modifications, and facilitates applications in gene therapy, drug discovery, and disease diagnosis by allowing for precise perturbation of genetic elements.
Implementation Method 1
a guide sequence hybridized to a target sequence within the target polynucleotide
Implementation Method 2
allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide
Data Source
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 include post mitotic cells which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing 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.