CRISPR-Cas Nickase Genome Editing via Guide RNA
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Solution Overview
Problem
Current genome editing technologies are not affordable, easy to set up, or scalable for targeting multiple positions within the eukaryotic genome, limiting their application in advanced biological and medical research.
Innovation Solution
The CRISPR/Cas system is used with a vector system that includes regulatory elements and guide sequences to direct sequence-specific binding of a CRISPR complex to target DNA sequences in eukaryotic cells, allowing for precise genome editing without the need for customized proteins, and can be optimized for expression in eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional genome editing technologies (designer zinc fingers, TALEs, homing meganucleases) are used, then targeted genome perturbations can be achieved, but the methods are not affordable, easy to set up, or scalable for multiple positions
Solution Approach 1:
The CRISPR-Cas9 system uses a single Cas9 enzyme that can be programmed to target multiple different genomic positions through exchange of guide RNA molecules. This universal platform replaces the need for custom protein engineering required by zinc finger nucleases and TALEs, making the system scalable and affordable for multiplexed genome editing applications
Solution Approach 2:
The invention uses RNA copies (guide RNA) to direct the Cas9 enzyme to specific DNA targets, rather than requiring custom protein sequences for each target site. The guide RNA can be easily synthesized and exchanged to program Cas9 for different genomic locations, dramatically simplifying the workflow compared to protein-based approaches
2Productivity
If CRISPR-Cas system is used for genome editing, then scalability and ease of use improve, but off-target effects and deleterious effects may occur
Solution Approach 1:
The patent employs high-fidelity Cas9 variants with modified catalytic domains that enhance discrimination between on-target and off-target sequences. The guide RNA is designed with optimized seed regions and mismatch tolerance parameters to improve local binding specificity at the target site while maintaining editing efficiency
Solution Approach 2:
The system uses nickase variants of Cas9 (with one catalytic domain inactivated) that create single-strand nicks instead of double-strand breaks. By using two guide RNAs targeting opposite strands, the system achieves double-strand breaks only when both guides bind correctly, significantly reducing off-target effects through a 'double-check' mechanism
3Reliability
If customized proteins are generated to target specific sequences, then sequence-specific binding is achieved, but the process becomes more complex and less affordable
Solution Approach 1:
The invention replaces the mechanical protein-DNA recognition system with an RNA-DNA hybridization system. Instead of engineering custom proteins with specific DNA-binding domains, the system uses guide RNA sequences that base-pair with target DNA, leveraging the well-understood and easily manipulated RNA-DNA hybridization chemistry
Solution Approach 2:
The guide RNA acts as an intermediary between the Cas9 enzyme and the target DNA sequence. The Cas9 enzyme provides the catalytic function while the guide RNA provides the sequence-specific recognition, separating the functions and allowing independent optimization of each component
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 simplifies genome editing, accelerates the mapping of genetic factors associated with biological functions and diseases, and enhances the scalability and affordability of genome engineering techniques.
Implementation Method 1
the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence
Implementation Method 2
CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence Manipulation
Data Source
AI summary
The invention provides for systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. 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 and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/Cas system.