Protected Guide RNA Structure for Specific CRISPR Targeting
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Solution Overview
Problem
Current genome-editing technologies, such as designer zinc fingers and TALEs, require customized proteins for specific sequence targeting and are not scalable or cost-effective for multiple positions within the eukaryotic genome, limiting their application in genome engineering and biotechnology.
Innovation Solution
The CRISPR-Cas9 system is optimized with guide RNAs that enhance specificity and protect against exonuclease activity, using a protected guide RNA (pgRNA) with a protector sequence and tracr mate sequence to target specific DNA sequences, reducing off-target effects and improving efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If customized proteins are used for sequence targeting, then specificity is improved, but device complexity and cost increase
Solution Approach 1:
The patent uses guide RNA molecules as simple copies or substitutes for complex customized proteins. Instead of designing unique proteins for each target sequence, the system uses a standardized Cas9 enzyme guided by programmable RNA sequences that can be easily synthesized and modified, dramatically simplifying the system while maintaining targeting specificity
Solution Approach 2:
The Cas9 enzyme serves as a universal platform that can target multiple different DNA sequences by simply changing the guide RNA molecule. This single enzyme performs the function of what would otherwise require multiple different customized proteins, reducing device complexity while maintaining the ability to achieve specific sequence targeting
2Device complexity
If guide RNA is used for targeting, then device complexity is reduced, but susceptibility to exonuclease activity increases
Solution Approach 1:
The patent creates a composite guide RNA structure by fusing the guide sequence with a tracrRNA sequence. This composite structure forms a stable duplex that protects the guide RNA from exonuclease degradation while maintaining its ability to direct Cas9 to the target sequence. The combination of guide RNA and tracrRNA creates a more robust molecule that retains functionality despite the added complexity of protection mechanisms
3Ease of operation
If standard guide RNA is used, then ease of operation is improved, but off-target effects increase
Solution Approach 1:
The patent modifies the guide RNA structure by adding the tracrRNA fusion and protecting against exonuclease activity. These parameter changes in the RNA molecule's stability and structure enhance the precision of target recognition and reduce off-target effects, while the system remains as easy to use as standard guide RNA since the protected guide RNA is supplied as a ready-to-use component
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 Cas9 enzyme programmed by a short RNA molecule, enhancing specificity and reducing off-target effects, thereby accelerating the cataloging and 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 eukaryotic cell
Implementation Method 2
the secondary structure can protect against exonuclease activity
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, wherein the guide sequence is modified by secondary structure to increase the specificity of the CRISPR-Cas system and whereby the secondary structure can protect against exonuclease activity and allow for 5′ additions to the guide sequence.