CRISPR-Cas9 Genome Editing via Programmable RNA
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Solution Overview
Problem
Current genome editing technologies, such as designer zinc fingers and TALEs, are not scalable, affordable, or easily adaptable for targeting multiple positions within the eukaryotic genome, limiting their applicability in reverse engineering genetic variations and synthetic biology applications.
Innovation Solution
The CRISPR/Cas system is employed, where a single Cas enzyme is programmed by a short RNA molecule to target specific DNA sequences, allowing for efficient and precise genome editing without the need for customized proteins, and is optimized for expression in eukaryotic cells, including human cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If customized proteins (designer zinc fingers, TALEs) are used for genome targeting, then specific DNA sequences can be targeted, but the system is not scalable, affordable, or easily adaptable for targeting multiple positions
Solution Approach 1:
The patent employs a single Cas9 enzyme that can be programmed by different guide RNAs to target multiple DNA sequences throughout the genome. This universal system replaces the need for customized proteins for each target site, enabling scalable and adaptable genome editing across multiple positions while maintaining targeting precision
Solution Approach 2:
The invention uses RNA molecules as programmable guides that can be easily synthesized and modified to match different DNA target sequences. Instead of creating new proteins for each target, the system copies the targeting function through programmable RNA sequences that direct the same Cas9 enzyme to different genomic locations
2Adaptability or versatility
If a single Cas enzyme is programmed by short RNA to target specific DNA sequences, then scalability and adaptability improve, but the system complexity increases
Solution Approach 1:
The system separates the genome editing function into two distinct components: a universal Cas9 enzyme that performs the cutting function, and programmable guide RNAs that provide targeting specificity. This segmentation allows the complex targeting function to be simplified into modular elements where the enzyme remains constant and only the RNA needs to be changed for different targets
Solution Approach 2:
The guide RNA acts as an intermediary molecule that bridges the Cas9 enzyme and the target DNA sequence. This intermediary carries the targeting information in a simple, programmable format, reducing the complexity of directly programming the enzyme itself while maintaining precise targeting capability
3Adaptability or versatility
If CRISPR-Cas system is optimized for expression in eukaryotic cells, then applicability to human cells improves, but the need for codon optimization and engineering increases
Solution Approach 1:
The patent optimizes the CRISPR-Cas system for eukaryotic expression by modifying parameters such as codon usage to match human cell preferences, optimizing RNA structure and stability, and adjusting protein expression levels. These parameter changes enable efficient function in eukaryotic cells while following established molecular biology optimization protocols
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, enhances the ability to map genetic factors associated with biological functions and diseases, and provides a robust and scalable method for altering gene expression, thereby accelerating the discovery of genetic insights and biotechnological advancements.
Implementation Method 1
a guide RNA that targets the DNA molecule encoding the gene product, whereby the guide RNA targets the DNA molecule
Implementation Method 2
the Cas protein cleaves the DNA molecule encoding the gene product
Data Source
AI summary
The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system.