CRISPR Guide RNA Delivery for Specific Genome Editing
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Solution Overview
Problem
Current genome-editing technologies, such as designer zinc fingers and TALEs, are not cost-effective, scalable, and difficult to set up for targeting multiple positions within the eukaryotic genome, necessitating the development of more affordable and efficient genome engineering tools.
Innovation Solution
The CRISPR-Cas system is utilized with a guide RNA to target specific DNA sequences, employing Cas9 enzymes with improved specificity and smaller size, and chimeric enzymes to enhance targeting, along with methods for optimizing delivery and reducing toxicity, enabling precise genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If designer zinc fingers or TALEs are used for genome editing, then specific DNA sequences can be targeted, but the cost is high and scalability is limited
Solution Approach 1:
The CRISPR-Cas9 system employs a universal platform where a single Cas9 enzyme can be programmed to target any DNA sequence by changing only the guide RNA sequence. This multi-functional approach allows the same enzymatic machinery to edit multiple different genomic locations, dramatically improving scalability compared to protein-based methods that require custom engineering for each target site.
Solution Approach 2:
The invention changes the key parameter from protein sequence (in zinc fingers and TALEs) to RNA sequence (in guide RNA). This parameter change enables simpler, faster, and cheaper modification of target specificity, as RNA sequences can be synthesized and exchanged without complex protein engineering, thereby resolving the scalability and cost issues.
2Measurement precision
If designer zinc fingers or TALEs are used for genome editing, then specific DNA sequences can be targeted, but setup complexity is high
Solution Approach 1:
The CRISPR system segments the targeting function from the enzymatic activity. The Cas9 enzyme remains constant while the guide RNA is segmented and can be independently designed for each target. This separation simplifies setup because only the small guide RNA needs to be customized for each target, rather than engineering entire protein complexes as required by zinc fingers and TALEs.
Solution Approach 2:
The guide RNA acts as a simple informational copy or blueprint that directs the Cas9 enzyme to the target sequence. This copying mechanism replaces complex protein-protein interactions required in zinc finger and TALE systems, dramatically reducing setup complexity while maintaining targeting precision.
3Ease of operation
If standard CRISPR-Cas9 is used for genome editing, then ease of operation is improved, but off-target effects increase
Solution Approach 1:
The invention introduces high-specificity Cas9 variants with modified local properties at the DNA binding interface. These engineered Cas9 proteins have altered amino acid sequences that enhance discrimination between on-target and off-target sequences, thereby reducing harmful off-target effects while preserving the ease of operation provided by the CRISPR system.
Solution Approach 2:
The patent employs composite Cas9 proteins that combine wild-type Cas9 domains with engineered specificity-enhancing domains or mutations. These composite enzymes integrate the beneficial properties of ease of use from wild-type Cas9 with the high specificity required to minimize off-target effects.
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 by providing a cost-effective, scalable, and versatile method for modifying nucleic sequences across various cell types and tissues, with applications in gene editing, therapy, drug discovery, and disease diagnosis.
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 the 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 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.