CRISPR-Cas Guide RNA Design for Scalable Genome Editing
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Solution Overview
Problem
Current genome editing technologies are not affordable, easy to set up, or scalable for targeting multiple positions within the eukaryotic genome, limiting their application in advanced synthetic biology and medical applications.
Innovation Solution
The CRISPR-Cas system, which uses a single Cas enzyme programmed by a short RNA molecule to recognize specific DNA targets, is integrated into genome sequencing techniques, enabling efficient and versatile genome editing by forming a CRISPR complex with a guide sequence hybridized to a target sequence, linked to a tracr mate sequence that hybridizes with a tracr sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If genome editing is performed using traditional methods (zinc fingers, TALEs, meganucleases), then targeted genome perturbation can be achieved, but the technology is not affordable, easy to set up, or scalable for multiple positions
Solution Approach 1:
The CRISPR system divides the genome targeting function into two separable components: a reusable Cas9 enzyme and interchangeable guide RNA molecules. Each guide RNA can be independently designed to target different genomic sequences, allowing easy reprogramming of the system for multiple targeting positions without redesigning the entire system.
Solution Approach 2:
A single Cas9 enzyme can perform multiple genome editing functions by accepting different guide RNA sequences. The Cas9 protein serves as a universal platform that can be programmed to target any DNA sequence containing the appropriate PAM motif, enabling broad adaptability across multiple genomic locations and applications.
2Manufacturing precision
If CRISPR-Cas system is used for genome editing, then targeting specificity is enhanced, but delivery and activity of CRISPR enzymes need improvement
Solution Approach 1:
The system separates the large Cas9 enzyme into deliverable fragments or uses alternative delivery methods (mRNA, protein transfection) to overcome size limitations of viral vectors. This segmentation allows the enzyme to be delivered more efficiently while maintaining its genome editing functionality and targeting specificity.
Solution Approach 2:
The patent employs viral vectors as intermediary carriers to deliver CRISPR components into cells. The viral delivery system acts as a mediator that protects the CRISPR components during transport and facilitates their entry into target cells, thereby improving delivery efficiency while preserving the enzyme's targeting capability.
3Adaptability or versatility
If multiple guide sequences are used to target multiple positions, then versatility increases, but system complexity increases
Solution Approach 1:
Multiple guide RNA sequences are combined into a single polycistronic transcript under the control of one promoter. This merging allows simultaneous expression of multiple guide RNAs from a single genetic construct, enabling multi-position targeting while simplifying the delivery system and reducing the number of separate components that need to be managed.
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 allows for broad applications in gene editing, therapy, disease modeling, and drug discovery by effectively modifying polynucleotides in various cell types and tissues, reducing toxicity and improving delivery and activity of CRISPR enzymes.
Implementation Method 1
a guide sequence hybridized to a target sequence within the target polynucleotide
Implementation Method 2
the guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence
Data Source
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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 enable genome engineering in an organism to recapitulate the genetic complexity of disease or a condition and further interrogate gene function.