RNA-Guided Nuclease Guide RNA Design for Specific Genome Editing
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Solution Overview
Problem
Existing genome editing methods, such as meganucleases and TALENs, are costly and inefficient for generating target-specific chimeric nucleases, while RNA-guided nucleases like CRISPR-Cas systems require complex guide RNA production.
Innovation Solution
Compositions and methods utilizing RNA-guided nuclease (RGN) polypeptides, CRISPR RNAs, and guide RNAs for specific binding and editing of target sequences, including homology-directed repair and base editing, with polynucleotides encoding RGN polypeptides having high sequence identity to specific amino acid sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If chimeric nucleases (meganucleases, zinc finger fusion proteins, TALENs) are engineered for each target sequence, then sequence-specific DNA-binding capability is achieved, but production cost and time increase significantly
Solution Approach 1:
The patent employs a universal nuclease domain (Cas9, Cas12a, or Cas13) that can recognize and bind to any target sequence when paired with an appropriately designed guide RNA. This single nuclease engine serves multiple functions across different target sequences, eliminating the need to engineer different nuclease proteins for each target, thereby dramatically reducing production cost and time while maintaining sequence-specific binding capability
Solution Approach 2:
The patent introduces guide RNA as an intermediary molecule that mediates between the universal nuclease and the target DNA sequence. The guide RNA contains a spacer sequence complementary to the target sequence, enabling the nuclease to achieve sequence-specific binding without requiring engineering of the nuclease protein itself. This intermediary approach resolves the contradiction by decoupling the universal binding mechanism from the sequence-specific recognition function
2Productivity
If CRISPR-Cas systems are used for genome editing, then editing efficiency improves, but guide RNA production complexity increases
Solution Approach 1:
The patent merges the crRNA and tracrRNA into a single synthetic guide RNA molecule that contains both the spacer sequence (for target recognition) and the scaffold sequence (for Cas protein binding). This consolidation simplifies guide RNA production by eliminating the need to assemble multiple separate RNA components, reducing production complexity while maintaining the high editing efficiency of the CRISPR-Cas system
Solution Approach 2:
The guide RNA is segmented into functional domains: a spacer sequence (20 nucleotides) for target sequence complementarity and a scaffold sequence for Cas protein interaction. This segmentation allows for modular design where only the spacer needs to be customized for each target, while the scaffold remains constant, simplifying production by allowing reuse of the standardized scaffold portion across different applications
3Productivity
If double-stranded breaks are introduced for genome editing, then mutation introduction capability improves, but risk of off-target effects increases
Solution Approach 1:
The patent employs nickases (nucleases with single-strand cutting activity) instead of full double-strand breaking nucleases. By introducing nicks (single-strand breaks) on either the top or bottom strand, the system achieves sufficient editing capability through nick repair mechanisms while dramatically reducing the risk of off-target effects and chromosomal abnormalities associated with double-strand breaks. This partial action approach maintains productivity while minimizing harmful effects
Solution Approach 2:
The patent uses different nuclease variants with distinct cutting specificities: some cut only the top strand, others cut only the bottom strand, and some require specific PAM sequences. This local differentiation in cutting behavior allows for tailored editing approaches where the nuclease activity is precisely localized to the intended target site with enhanced specificity, reducing off-target effects while maintaining the ability to introduce mutations at the desired location
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
Provides efficient and cost-effective genome editing by enabling precise binding and modification of target sequences, including double-strand breaks and base editing, with improved specificity and reduced production costs.
Implementation Method 1
a guide RNA (gRNA) capable of hybridizing to the target DNA sequence
Implementation Method 2
the RGN polypeptide is capable of binding a target DNA sequence in an RNA-guided sequence specific manner when bound to a guide RNA (gRNA) capable of hybridizing to the target DNA 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 (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.