RNA-Guided Nuclease Specificity via Dual-RNA Intermediary
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Solution Overview
Problem
Current genome editing tools, such as RNA-guided nucleases, face limitations in specificity and efficiency for targeted modifications, particularly in achieving precise gene editing in therapeutic and research applications, especially in mammals and crop plants.
Innovation Solution
Development of RNA-guided nuclease systems comprising CRISPR-Cas proteins, guide RNAs, and deaminase polypeptides that enable targeted binding, cleavage, and base editing of nucleic acid sequences through non-homologous end joining or homology-directed repair, allowing for precise modification of genomic loci in eukaryotic and prokaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If RNA-guided nucleases are used for targeted genome editing, then the ability to introduce specific mutations is improved, but the specificity and efficiency for targeted modifications remain limited
Solution Approach 1:
The invention divides the guide RNA into separate crRNA and tracrRNA components that must assemble together to form a functional complex with the Cas9 nuclease. This segmentation increases specificity because both RNA components must simultaneously bind to the target sequence, reducing off-target effects while maintaining editing precision
Solution Approach 2:
The invention introduces a dual-RNA intermediary system (crRNA-tracrRNA complex) that mediates between the Cas9 nuclease and the target DNA sequence. This intermediary provides an additional layer of sequence recognition and verification, enhancing both the precision and reliability of targeted genome editing
2Adaptability or versatility
If chimeric nucleases with engineered DNA-binding domains are generated for each target sequence, then sequence-specific targeting is achieved, but the cost and complexity increase significantly
Solution Approach 1:
The invention creates a universal Cas9 nuclease platform that can target any DNA sequence by simply changing the guide RNA sequence. The Cas9 protein itself remains unchanged and reusable, while the programmable crRNA-tracrRNA complex provides the sequence-specific binding capability, dramatically reducing complexity compared to generating new chimeric nucleases for each target
Solution Approach 2:
The invention uses RNA copies (guide RNAs) to encode the targeting information instead of requiring unique protein constructions for each target. The sequence-specificity is copied into the RNA molecule rather than being built into a new protein each time, simplifying the adaptation process for different genomic targets
3Productivity
If RNA-guided nucleases create double-stranded breaks for genome editing, then targeted mutations can be introduced, but the risk of off-target effects and genomic instability increases
Solution Approach 1:
The invention employs a dynamic, modular guide RNA system where crRNA and tracrRNA can be easily reconfigured to match different target sequences. This dynamic adaptability allows for optimized targeting that maximizes on-efficiency while minimizing off-target effects through careful design of the RNA-DNA hybridization parameters
Solution Approach 2:
The dual-RNA system provides built-in feedback mechanisms for target verification. Both crRNA and tracrRNA must simultaneously recognize and bind to their respective target sequences, creating a feedback loop that confirms correct target identification before Cas9 cleavage occurs, thereby reducing off-target effects
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
Enables precise and efficient modification of genomic sequences, facilitating therapeutic applications in mammals and the introduction of beneficial traits in plants, with improved specificity and reduced costs compared to earlier methods.
Implementation Method 1
complexing the nucleases with guide RNA that specifically hybridizes with a particular target sequence
Implementation Method 2
Such RNA-guided nucleases can be used to edit genomes through the introduction of a sequence-specific, double-stranded break
Implementation Method 3
base editing
Data Source
AI summary
Compositions and methods for binding to a target sequence of interest are provided. Compositions include fusion proteins between DNA binding proteins or protein domains and nucleic acid modifying proteins or protein domains. The compositions find use in cleaving or modifying a target sequence of interest, visualization of a target sequence of interest, and modifying the expression of a sequence of interest. Compositions comprise RNA-guided nuclease polypeptides, CRISPR RNAs, trans-activating CRISPR RNAs, guide RNAs, deaminases, and nucleic acid molecules encoding the same. Vectors and host cells comprising the nucleic acid molecules are also provided. Further provided are CRISPR systems for binding a target sequence of interest, wherein the CRISPR system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs. Also provided are deaminases which may be fused to a DNA-binding polypeptide and may be useful for gene editing.