Engineered CRISPR Guide Architectures for Precise Genome Targeting
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Solution Overview
Problem
Current genome-editing techniques, such as designer zinc fingers and TALEs, are costly, complex, and not scalable for targeting multiple positions within eukaryotic genomes, necessitating a more affordable and efficient method for precise genome perturbation.
Innovation Solution
The CRISPR/Cas system is utilized with engineered guide RNA (sgRNA) architectures that can target specific DNA sequences using a single Cas enzyme, allowing for simplified and scalable genome editing by modifying the sgRNA structure to enhance specificity and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional genome-editing techniques (designer zinc fingers, TALEs) are used, then precise genome targeting is achieved, but the system becomes costly and complex with limited scalability
Solution Approach 1:
The patent uses a single Cas9 enzyme that can be programmed with different guide RNAs to target different genomic locations, effectively copying the targeting function across multiple sites without requiring multiple different enzyme systems. This reduces complexity while maintaining precision
Solution Approach 2:
The Cas9 enzyme is designed as a universal tool that can target multiple positions in the eukaryotic genome by simply changing the guide RNA sequence, making the system scalable and affordable for multiplexed genome editing applications
2Measurement precision
If traditional genome-editing techniques are used, then precise genome targeting is achieved, but affordability and scalability are reduced
Solution Approach 1:
Instead of manufacturing multiple different complex protein systems, the patent copies the targeting capability through programmable guide RNAs that direct a single Cas9 enzyme to different locations, significantly reducing manufacturing costs and improving scalability
Solution Approach 2:
The system allows easy reprogramming by changing the guide RNA sequence parameters, enabling rapid adaptation to different target sites without requiring complex protein engineering, thus improving affordability and scalability
3Productivity
If a single Cas enzyme is used with programmable guide RNA, then scalability and affordability are improved, but guide RNA architecture optimization is needed to maintain precision
Solution Approach 1:
The patent optimizes specific local regions of the guide RNA architecture (such as the seed region and terminal loops) to enhance binding specificity and reduce off-target effects, while maintaining the overall simplicity and scalability of the single Cas9 system
Solution Approach 2:
The guide RNA architecture is designed with dynamic elements that allow for optimization of binding kinetics and specificity, enabling the system to maintain high precision while retaining the scalability advantages of the programmable approach
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 using a single Cas enzyme programmed by a short RNA molecule, enabling precise and affordable targeting of multiple genomic positions, accelerating the mapping of genetic factors associated with biological functions and diseases.
Implementation Method 1
a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest
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. In particular the present invention comprehends optimized functional CRISPR-Cas enzyme systems. In particular the present invention comprehends engineered new guide architectures to be used in optimized CRISPR-Cas enzyme systems.