Programmable CRISPR Guide Vectors for Scalable Genome Targeting
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Solution Overview
Problem
Current genome-editing techniques are costly, complex, and not scalable for efficient targeting of multiple positions within the eukaryotic genome, necessitating the development of affordable and easy-to-use systems for precise genome perturbation.
Innovation Solution
The CRISPR/Cas system is utilized with a programmable short RNA molecule to target specific DNA sequences, employing a vector system with regulatory elements and guide sequences to direct sequence-specific binding and cleavage, and optionally includes nuclear localization sequences for enhanced activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional genome-editing techniques (designer zinc fingers, TALEs, homing meganucleases) are used, then targeted genome perturbation is achieved, but the system becomes costly and complex
Solution Approach 1:
The patent uses RNA molecules as programmable guides that copy the target sequence information to direct the Cas enzyme to specific genomic locations, replacing complex protein-based targeting systems with simpler RNA-mediated recognition
Solution Approach 2:
A single Cas enzyme can be programmed by different RNA molecules to target multiple different genomic sequences, making the system universally applicable across various target sites without requiring different enzymes for each target
2Reliability
If traditional genome-editing techniques are used, then targeted genome perturbation is achieved, but the system becomes expensive and not scalable
Solution Approach 1:
The CRISPR-Cas system allows a single enzyme to perform multiple targeting functions by simply changing the RNA guide sequence, enabling scalable editing of multiple genomic positions without proportionally increasing system complexity or cost
Solution Approach 2:
The system changes the programmable parameter from protein structure design to RNA sequence composition, allowing rapid and inexpensive reconfiguration for different target sequences by simply altering the nucleotide sequence of the guide RNA
3Ease of operation
If CRISPR-Cas system is used without optimization, then genome editing is simplified, but efficiency and nuclear accumulation are reduced
Solution Approach 1:
The patent introduces a fusion protein comprising the Cas enzyme linked to a nuclear localization signal (NLS) that acts as an intermediary to facilitate transport of the CRISPR complex into the nucleus, thereby enhancing editing efficiency without complicating the overall system design
Solution Approach 2:
The CRISPR complex is engineered as a composite system combining the Cas enzyme, guide RNA, and nuclear localization signal into an integrated molecular assembly that maintains simplicity while improving functional performance
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, enabling efficient and scalable targeting of genetic elements, accelerating the mapping of genetic factors associated with diverse biological functions and diseases.
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
allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide
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.