Guide RNA and RGN Composition for Precise Genome Editing
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Solution Overview
Problem
Existing genome editing methods, such as meganucleases and TALENs, require costly and inefficient generation of chimeric nucleases for sequence-specific targeting, while RNA-guided nucleases like CRISPR-Cas systems offer a more efficient and cost-effective alternative for targeted genome editing and modification.
Innovation Solution
Compositions and methods utilizing RNA-guided nucleases (RGNs) with guide RNAs for sequence-specific binding and cleavage, including variants and fragments, enable targeted genome editing through non-homologous end-joining, homology-directed repair, and base editing, with applications in gene modification and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If RNA-guided nucleases are used to target specific genomic sequences, then editing precision is improved, but off-target effects still occur reducing reliability
Solution Approach 1:
The patent introduces an intermediary verification step by fusing the nuclease domain with a DNA-binding domain that recognizes the target sequence through a guide RNA. This intermediary recognition mechanism allows for more precise targeting by requiring both RNA-guided binding and DNA sequence matching, thereby reducing off-target effects while maintaining high editing precision at the intended genomic location.
Solution Approach 2:
The patent modifies the parameters of the nuclease system by using different guide RNA sequences with varying degrees of complementarity to the target site. By optimizing the guide RNA design and adjusting binding parameters, the system achieves higher specificity for the intended target while minimizing binding to off-target sequences, thus improving both editing precision and reliability.
2Productivity
If guide RNA is designed to target specific sequences, then targeting efficiency is improved, but system complexity increases due to multiple RNA components
Solution Approach 1:
The patent combines multiple RNA components (crRNA and tracrRNA) into a single fused guide RNA molecule. This merging simplifies the system by eliminating the need for separate RNA components while maintaining the targeting efficiency, as the fused guide RNA still contains all necessary elements for guide RNA-mediated target recognition and nuclease recruitment.
Solution Approach 2:
The patent creates a universal guide RNA design that can target multiple different genomic sequences by simply changing the spacer region sequence. This multi-functional guide RNA structure maintains high targeting efficiency across different targets while simplifying the overall system, as the same basic architecture can be reused for various editing applications without requiring entirely new components.
3Adaptability or versatility
If multiple repair pathways are utilized for genome modification, then editing versatility is improved, but control over specific modification outcomes becomes more difficult
Solution Approach 1:
The patent introduces a preliminary action by designing the guide RNA and nuclease system to create a specific type of double-strand break that preferentially directs cells toward a desired repair pathway. By controlling the characteristics of the break and the local genomic context, the system can bias toward either NHEJ or HDR outcomes, providing better control over modification precision while maintaining versatility through pathway selection.
Solution Approach 2:
Instead of trying to control which repair pathway the cell naturally chooses, the patent inverts the approach by using the guide RNA design and nuclease timing to actively direct the cell toward a specific pathway. This inversion of control strategy allows for more precise outcome control while maintaining the versatility of having multiple repair pathways available, as the system can be configured to favor either pathway as needed.
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 genome editing capabilities, enabling precise modification and detection of target sequences in various organisms, including mammals, through single- or double-strand breaks and modulation of gene expression.
Implementation Method 1
complexing the nucleases with guide RNA that specifically hybridizes with a particular target sequence
Implementation Method 2
introduce a sequence-specific, double-stranded break that is repaired via error-prone non-homologous end-joining (NHEJ)
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 (RGN) 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 RGN systems for binding a target sequence of interest, wherein the RGN system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs.