CRISPR-Cas Delivery for Precise Cleavage of Episomal HBV DNA
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Solution Overview
Problem
Current genome-editing techniques are not affordable, easy to set up, and scalable for targeting multiple positions within the eukaryotic genome, and there is a need for alternative systems to address persistent viral infections like HBV integration.
Innovation Solution
The CRISPR-Cas system is utilized with engineered guide RNAs and Cas9 enzymes, optimized for specific targeting and delivery, to modify target polynucleotides and reduce the abundance of episomal HBV DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current genome-editing techniques (designer zinc fingers, TALEs, homing meganucleases) are used to target specific sequences, then genome editing capability is achieved, but the system becomes complex, expensive, and difficult to scale for multiple positions
Solution Approach 1:
The CRISPR-Cas9 system employs a universal Cas9 enzyme that can be programmed to target any DNA sequence by simply changing the guide RNA sequence. This single enzyme system replaces the need for multiple different protein complexes (designer zinc fingers, TALEs, homing meganucleases), each requiring custom engineering for every target site. The guide RNA acts as a programmable address label, allowing the same Cas9 enzyme to precisely locate and edit different genomic positions without changing the enzyme itself, thus reducing system complexity while maintaining targeting precision.
Solution Approach 2:
The system changes the key parameter from protein sequence design to RNA sequence design. Instead of engineering complex protein-DNA recognition interfaces for each target, the system uses simple RNA-DNA base pairing rules where the guide RNA sequence directly complements the target DNA sequence. This parameter change from protein engineering to nucleic acid sequencing dramatically simplifies the system while preserving precise targeting capability.
2Measurement precision
If current genome-editing techniques are used, then targeted genome perturbation is achieved, but the methodology becomes expensive and not scalable
Solution Approach 1:
The CRISPR-Cas9 system enables multiplexed genome editing by allowing simultaneous introduction of multiple guide RNAs, each targeting different genomic positions. This universality allows researchers to edit multiple genes or regulatory elements in a single experiment, dramatically improving productivity and scalability compared to sequential editing with traditional methods. The system can target dozens of positions concurrently without increasing per-experiment complexity proportionally.
Solution Approach 2:
The system segments the genome editing function into two independent components: the Cas9 enzyme (cutting machine) and the guide RNA (address label). This segmentation allows the guide RNA to be easily replicated and modified for different targets while the Cas9 enzyme remains constant. Multiple guide RNAs can be synthesized independently and introduced together, enabling scalable parallel editing of multiple genomic locations without requiring proportional increases in experimental complexity or cost.
3Ease of operation
If CRISPR-Cas system is used for genome editing, then simplicity and scalability are improved, but delivery to specific cell types and tissues requires optimization
Solution Approach 1:
The patent employs delivery vehicles (viral vectors, liposomes, nanoparticles) as intermediaries to transport the CRISPR-Cas9 components into specific cell types and tissues. These intermediaries are engineered with cell-type specific targeting ligands or tropism modifications that enable selective delivery to desired tissues (e.g., liver for HBV treatment) while protecting the fragile Cas9 enzyme and guide RNA during transit. This intermediary approach maintains the simplicity of the CRISPR system while adding the necessary adaptability for in vivo applications.
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, enhances targeting specificity, and effectively reduces HBV persistence by directly cleaving the episomal form of the virus, offering broad applications in gene editing, therapy, and disease treatment.
Implementation Method 1
a guide sequence hybridized or hybridizable to a target sequence within the target polynucleotide
Implementation Method 2
CRISPR-Cas system... to modify target polynucleotides and reduce the abundance of episomal HBV DNA
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.