CRISPR Guide RNA Vector Design for Scalable Genome Targeting
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Solution Overview
Problem
Existing genome-editing techniques are costly, complex, and not scalable for targeting multiple positions within the eukaryotic genome, necessitating the development of affordable and easy-to-use systems for precise genome perturbation.
Innovation Solution
The CRISPR/Cas system is utilized with a programmable short RNA molecule to target specific DNA sequences, employing vector systems with regulatory elements and CRISPR enzymes like Cas9 for efficient genome editing, which can be optimized for eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing genome-editing techniques (designer zinc fingers, TALEs, homing meganucleases) are used, then targeted genome perturbation can be achieved, but the cost is high and the system complexity increases
Solution Approach 1:
The patent uses a simplified RNA copy (guide RNA) to direct the Cas enzyme to target sequences, replacing the need for complex protein-DNA recognition systems. The guide RNA contains the target sequence information, allowing the enzyme to be programmed through RNA rather than requiring complex customized protein structures.
Solution Approach 2:
The patent changes the recognition parameter from protein-DNA interaction (complex) to RNA-DNA hybridization (simpler). By using RNA guide molecules that base-pair with target DNA sequences, the system achieves specific targeting through well-understood nucleic acid pairing rules rather than complex protein structural recognition.
2Measurement precision
If existing genome-editing techniques are used, then specific genome positions can be targeted, but scalability to multiple positions is limited
Solution Approach 1:
The Cas enzyme serves as a universal platform that can target multiple different genome positions by simply changing the guide RNA sequence. The enzyme itself remains unchanged, while the programmable RNA guides direct it to various target sites, enabling multiplexed editing across multiple positions simultaneously.
Solution Approach 2:
The patent separates the targeting function (guide RNA) from the enzymatic function (Cas enzyme). This segmentation allows independent optimization and combination of multiple guide RNAs with a single Cas enzyme, enabling simultaneous targeting of multiple positions without requiring multiple different enzyme variants.
3Measurement precision
If customized proteins are generated for each target sequence, then specific binding can be achieved, but the ease of operation decreases
Solution Approach 1:
The guide RNA molecules are designed to be self-complementary and form hairpin structures that are automatically processed by the Cas enzyme complex. The system uses itself to generate the active guide RNA from precursor forms, reducing the need for complex external processing and setup procedures.
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 methodologies, enabling systematic cataloging and mapping of genetic factors associated with diverse biological functions and diseases, while avoiding deleterious effects.
Implementation Method 1
the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence... the guide sequence that is hybridized to the target sequence
Implementation Method 2
the tracr mate sequence that is hybridized to the tracr sequence
Implementation Method 3
a CRISPR enzyme comprising a nuclear localization sequence... drive accumulation of said CRISPR complex in a detectable amount in the nucleus
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.