Engineered CRISPR-Cas Endonucleases for Nucleic Acid Specificity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
There is a need for improved Class 2 CRISPR-Cas RNA-guided endonucleases that can efficiently target and modify specific DNA or RNA sequences with enhanced specificity and efficiency, particularly for applications in genome editing and nucleic acid detection.
Innovation Solution
Development of novel Class 2 Type II, Type V, and Type VI CRISPR-Cas RNA-guided proteins and systems, including engineered endonucleases and guide RNAs, which can specifically cleave target DNA or RNA sequences, and methods for their use in modifying or detecting nucleic acids, such as those encoding the RuvC or HEPN sequences with high sequence identity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If natural CRISPR-Cas systems are used, then the system is simple and easy to understand, but the specificity and efficiency for targeting nucleic acids are insufficient
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues in the RuvC and HEPN domains of Cas proteins to enhance nucleic acid binding affinity and cleavage efficiency. Specific mutations in catalytic residues and interface regions optimize the interaction between the endonuclease and target nucleic acid, thereby improving specificity without fundamentally changing the system architecture
Solution Approach 2:
The patent segments the CRISPR-Cas system into distinct functional modules: the guide RNA component for target recognition, the Cas protein scaffold for structural stability, and the RuvC/HEPN catalytic domains for nucleic acid cleavage. This modular approach allows independent optimization of each component's function while maintaining overall system simplicity
2Productivity
If existing Class 2 CRISPR-Cas endonucleases are used, then the system structure is minimal, but the efficiency for cleaving target DNA or RNA is insufficient
Solution Approach 1:
The patent optimizes cleavage efficiency by engineering specific amino acid substitutions in the catalytic domains that enhance the rate of phosphodiester bond hydrolysis. Mutations in the active site geometry and electrostatic environment of RuvC and HEPN domains accelerate nucleic acid cleavage kinetics while maintaining target specificity through preserved guide RNA complementarity requirements
Solution Approach 2:
The patent replaces the natural, less efficient cleavage mechanism with engineered catalytic domains that utilize optimized metal ion coordination and transition state stabilization. The modified RuvC and HEPN domains employ enhanced magnesium ion binding and improved catalytic residue positioning to achieve higher turnover rates compared to wild-type endonucleases
3Measurement precision
If the CRISPR-Cas system is engineered for higher specificity, then the targeting precision improves, but the complexity of designing and optimizing the system increases
Solution Approach 1:
The patent achieves enhanced targeting precision through site-directed mutagenesis of specific amino acid residues that contact the guide RNA and target nucleic acid. By optimizing hydrogen bonding networks, van der Waals contacts, and electrostatic interactions at the binding interface, the engineered endonucleases display improved discrimination between perfectly matched and mismatched targets
Solution Approach 2:
The patent creates simplified copyable variants of CRISPR-Cas systems with standardized genetic sequences for the guide RNA and Cas protein. These engineered systems can be easily replicated and transferred between different experimental contexts through standard molecular cloning techniques, reducing the practical complexity despite the underlying molecular optimizations
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
These novel CRISPR-Cas systems enable precise and efficient modification or detection of target nucleic acids, offering improved specificity and efficiency in genome editing and diagnostic applications, including the ability to target various types of nucleic acids like mRNA, tRNA, and viral sequences like SARS-CoV-2.
Implementation Method 1
The gRNA can be capable of hybridizing to a target sequence in a target DNA or RNA
Implementation Method 2
CRISPR-Cas proteins for RNA-guided nucleic acid cleavage
Data Source
AI summary
Provided herein are novel Class 2 Type II, Type V, type VI CRISPR-Cas RNA-guided endonucleases and systems comprising the same. Provided also are methods of making, and methods of use thereof. Exemplary methods of use include modifying target nucleic acids useful for therapeutic applications, and also include detecting targeting nucleic acids, useful for diagnostic applications.