RNA-Guided Nucleases with Guide RNA for Targeted Genome Editing
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Solution Overview
Problem
Existing genome editing methods, such as meganucleases and TALENs, are costly and inefficient for targeted sequence modification, while RNA-guided CRISPR systems require complex chimeric nucleases for each target sequence.
Innovation Solution
Compositions and methods using RNA-guided nucleases (RGNs) with guide RNAs for targeted genome editing, including CRISPR systems, enable specific binding and modification of target sequences through non-homologous end-joining or homology-directed repair, with variants capable of cleavage or expression modulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If RNA-guided CRISPR systems are used for targeted genome editing, then productivity and cost-effectiveness are improved, but device complexity increases due to requirement of complex chimeric nucleases for each target sequence
Solution Approach 1:
The patent applies universality by developing a single RNA-guided nuclease system (CRISPR-Cas) that can target multiple different genomic sequences through exchange of guide RNAs, eliminating the need to design and construct different chimeric nucleases for each target. The Cas nuclease remains constant while only the guide RNA needs to be changed, making the system universally applicable across numerous targets.
Solution Approach 2:
The patent segments the targeting function from the nuclease function. The guide RNA contains the target-specific sequence information and directs the nuclease to the correct location, while the Cas nuclease provides the cleavage function. This segmentation allows independent optimization of each component and simplifies the overall system compared to integrated chimeric nucleases.
2Manufacturing precision
If chimeric nucleases are engineered for each target sequence, then manufacturing precision is improved, but ease of manufacture deteriorates due to costly and inefficient production processes
Solution Approach 1:
The patent uses guide RNAs as information carriers that can be easily synthesized and replicated. Instead of engineering entire chimeric nuclease proteins for each target, the target-specific information is copied into RNA sequences that guide the constant Cas nuclease to the correct location, dramatically simplifying the manufacturing process.
Solution Approach 2:
The patent changes the parameter that needs to be modified for each target from the protein sequence (in chimeric nucleases) to the RNA sequence (in guide RNAs). This parameter change makes the system much easier to manufacture because RNA synthesis is simpler, faster, and more cost-effective than protein engineering and expression.
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
RGNs provide efficient and cost-effective targeted genome editing, enabling precise modification and detection of sequences, as well as modulation of gene expression, in various organisms.
Implementation Method 1
complexing the nucleases with guide RNA that specifically hybridizes with a particular target sequence
Data Source
AI summary
Compositions and methods for binding to a target sequence of interest are provided. 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, 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.