CRISPR Guide RNA Design for Scalable Genome Editing
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Solution Overview
Problem
Current genome editing technologies, such as CRISPR, require customized proteins for specific sequence targeting, which can be complex and costly, limiting their scalability and ease of use for targeting multiple positions in the eukaryotic genome.
Innovation Solution
The CRISPR/Cas system uses a single Cas enzyme programmed by a short RNA molecule to recognize specific DNA targets, eliminating the need for customized proteins and simplifying the methodology for genome editing by enabling sequence-specific binding and cleavage without DNA strand cleavage activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If customized proteins are used for sequence targeting, then targeting specificity is improved, but device complexity and cost increase
Solution Approach 1:
The patent uses RNA molecules as simple copies that can be easily synthesized and exchanged to target different genomic sequences, replacing the need for complex customized protein designs. The RNA guide sequences serve as information carriers that direct the Cas enzyme to specific targets through base pairing, significantly reducing system complexity while maintaining targeting precision.
Solution Approach 2:
The patent changes the targeting mechanism from protein-based recognition to RNA-based recognition. By altering the fundamental parameter of what molecule performs sequence-specific binding (from customized proteins to RNA guides), the system achieves both high targeting specificity and reduced complexity, as RNA sequences can be easily designed and synthesized for different targets.
2Measurement precision
If customized proteins are generated for each target, then targeting accuracy is improved, but ease of manufacture and scalability worsen
Solution Approach 1:
Instead of generating customized proteins for each target, the system uses RNA guide sequences that can be rapidly synthesized and exchanged. These RNA copies contain the sequence information needed to target specific genomic locations, making the system much easier to manufacture and scale to multiple targets.
Solution Approach 2:
The Cas enzyme serves as a universal platform that can target multiple different genomic sequences by exchanging RNA guide molecules. This multi-functional approach allows a single enzyme to perform numerous targeting tasks, greatly improving ease of manufacture and scalability compared to generating customized proteins for each target.
3Adaptability or versatility
If multiple positions in the eukaryotic genome are targeted, then versatility is improved, but device complexity and cost increase
Solution Approach 1:
The system achieves multi-position targeting capability through a universal Cas enzyme platform that can be directed to different genomic locations by exchanging RNA guide sequences. Each RNA guide is designed to target a specific position, but the same Cas enzyme can perform all targeting functions, greatly simplifying the system compared to using multiple customized proteins.
Solution Approach 2:
The system uses multiple RNA guide sequence copies, each targeting a different genomic position. These RNA copies are simple molecules that can be easily synthesized and combined with the Cas enzyme, allowing versatile multi-position targeting without increasing system complexity.
4Measurement precision
If customized proteins are used, then sequence recognition precision is improved, but productivity and scalability worsen
Solution Approach 1:
The system replaces customized proteins with RNA guide sequence copies that can be rapidly synthesized and exchanged. This copying approach maintains sequence recognition precision through complementary base pairing while dramatically improving productivity and scalability, as RNA molecules can be produced much more efficiently than customized proteins.
Solution Approach 2:
The patent changes the molecular basis of sequence recognition from protein-DNA interactions to RNA-DNA hybridization. This parameter change enables both high recognition precision and improved scalability, as RNA sequences can be easily designed, synthesized, and scaled for multiple targets without the complexities of protein engineering.
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 and accelerates the ability to catalog and map genetic factors associated with biological functions and diseases, offering a robust and scalable method for genome engineering with reduced complexity and cost.
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
a single Cas enzyme can be programmed by a short RNA molecule to recognize a specific DNA target... including such sequences enhances activity of the system, especially as to targeting nucleic acid molecules in the nucleus
Data Source
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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.