CRISPR-Cas Vector Systems Using Guide RNA for Scalable Genome Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genome-editing techniques are costly, complex, and not scalable for targeting multiple positions within the eukaryotic genome, necessitating a more affordable and efficient method for precise genome perturbation.
Innovation Solution
The CRISPR/Cas system uses a single Cas enzyme programmed by a short RNA molecule to target specific DNA sequences, combined with vector systems and regulatory elements for efficient genome editing without requiring customized proteins.
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 DNA sequences, then sequence-specific binding is achieved, but the system requires customized proteins for each target and becomes costly and complex
Solution Approach 1:
The Cas9 enzyme serves as a universal platform that can target multiple different DNA sequences through programming with different guide RNAs. This single enzyme performs the function of multiple customized proteins, eliminating the need to design and produce different protein complexes for each target sequence.
Solution Approach 2:
The guide RNA acts as a programmable copy or template that directs the Cas9 enzyme to specific DNA sequences. Instead of creating custom proteins for each target, the system uses RNA molecules that can be easily synthesized and exchanged to program the same Cas9 enzyme for different targets.
2Adaptability or versatility
If current genome-editing techniques are used to target multiple positions within the eukaryotic genome, then comprehensive genome perturbation is achieved, but the methodology becomes not scalable and difficult to set up
Solution Approach 1:
The CRISPR-Cas system provides a universal platform where the same Cas9 enzyme and basic machinery can be programmed to target multiple positions in the genome by simply changing the guide RNA sequences, enabling scalable and versatile genome-wide screening and editing applications.
Solution Approach 2:
The system separates the targeting function into modular guide RNA components that can be independently designed and combined with the Cas9 enzyme. This segmentation allows easy reconfiguration for different targets without redesigning the entire system.
3Manufacturing precision
If current genome-editing techniques are implemented, then precise genome perturbation is achieved, but the cost and complexity increase significantly
Solution Approach 1:
The system uses easily synthesized RNA molecules as programmable guides instead of requiring complex protein engineering for each application. This copying approach with RNA templates dramatically reduces the cost and technical barrier for implementing precise genome editing.
Solution Approach 2:
The guide RNAs are inexpensive, easily synthesized molecules that can be discarded and replaced for different applications. This replaces the need for expensive, custom-engineered proteins that require complex production and purification processes.
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, accelerates the mapping of genetic factors associated with diverse biological functions and diseases, and enhances the scalability and affordability of genome engineering.
Implementation Method 1
the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence... wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) 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
the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence
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.