CRISPR-Cas9 Genome Editing via Guide RNA Segmentation
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Solution Overview
Problem
Current genome engineering technologies lack affordable, easy-to-set-up, scalable, and versatile methods for precise targeting of multiple positions within the eukaryotic genome, limiting their applications in genome engineering and biotechnology.
Innovation Solution
Development of novel DNA or RNA-targeting systems using Type V CRISPR-Cas loci effector proteins, such as C2c1 or C2c3, which form complexes with nucleic acid components to induce modifications, including strand breaks, at specific target loci within genomes, enabling efficient genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional genome editing technologies (zinc fingers, TALEs, meganucleases) are used, then targeted genome perturbation is achieved, but the system complexity and cost increase, reducing scalability and ease of setup
Solution Approach 1:
The CRISPR-Cas system divides genome editing into separate functional components: guide RNA for targeting and Cas9 protein for cutting. This segmentation allows independent optimization of each component and simplifies the overall system compared to conventional methods that require designing entire protein structures for each target site.
Solution Approach 2:
The CRISPR-Cas9 system provides universal applicability through the use of guide RNA that can be designed to target any genomic location. A single Cas9 protein can perform multiple editing functions (knockout, knockin, regulation) by changing only the guide RNA sequence, making the system more versatile and scalable than conventional methods that require different proteins for different applications.
2Manufacturing precision
If conventional genome editing methods are used, then genome targeting is achieved, but affordability and ease of setup are reduced, limiting accessibility
Solution Approach 1:
The patent extracts the targeting function from the cutting function by using separate guide RNA and Cas9 protein components. This extraction simplifies the system setup process as researchers only need to order pre-assembled CRISPR kits rather than designing and expressing entire protein constructs, significantly improving ease of setup and affordability.
Solution Approach 2:
The guide RNA can be easily synthesized and discarded after use, while the Cas9 protein serves as a reusable enzyme. This approach reduces costs compared to conventional methods that require expensive, complex protein expressions for each target site, making the technology more accessible to smaller labs and institutions.
3Productivity
If CRISPR-Cas systems are used for genome editing, then scalability is improved, but off-target effects and lack of control over modification type increase
Solution Approach 1:
The system provides local control by allowing researchers to design guide RNA specific to particular genomic locations while using a universal Cas9 protein. This local quality approach enables precise targeting at desired sites while maintaining the ability to control the type of modification (knockout, knockin, regulation) through careful design of the guide RNA and delivery method.
Solution Approach 2:
The CRISPR-Cas system offers dynamic control through the ability to switch between different guide RNA sequences to target different locations, and to modulate the duration and intensity of editing by controlling Cas9 expression timing and levels. This dynamic adaptability allows researchers to optimize for either scalability or precision depending on the specific application needs.
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
These systems provide robust and versatile tools for precise genome editing, allowing for targeted modifications in various cell types, including non-dividing cells, with minimal off-target effects, thereby enhancing genome engineering capabilities.
Implementation Method 1
the effector protein induces the modification of the sequences associated with or at the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break.
Data Source
AI summary
The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.