Guide RNA-Directed Nucleases for Precise HDR and Base Editing
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Solution Overview
Problem
Existing genome editing methods, such as meganucleases, zinc finger fusion proteins, and TALENs, require costly and inefficient generation of chimeric nucleases for sequence-specific targeting, while RNA-guided nucleases like CRISPR-Cas systems face challenges in precision and efficiency, particularly in base editing and homology-directed repair.
Innovation Solution
Compositions and methods utilizing RNA-guided nuclease (RGN) polypeptides, CRISPR RNAs, and guide RNAs for sequence-specific binding, cleavage, and modification, including homology-directed repair and base editing, with polynucleotides encoding RGN polypeptides having high sequence identity to specific amino acid sequences, and vectors for expression in eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If chimeric nucleases (meganucleases, zinc finger fusion proteins, TALENs) are used for targeted genome editing, then sequence-specific targeting capability is achieved, but the cost and efficiency of generating custom nucleases for each target sequence becomes prohibitively high
Solution Approach 1:
The patent uses guide RNA molecules as programmable templates that specify target sequences through base pairing, eliminating the need to design and construct new protein domains for each target. The guide RNA acts as a simple, cheap copyable template that directs the Cas nuclease to the desired genomic location, replacing the complex chimeric nuclease design process
Solution Approach 2:
The invention changes the programmable parameter from protein sequence (in chimeric nucleases) to RNA sequence (in guide RNAs). This parameter change allows for simpler, more efficient generation of target-specific editors by merely changing the guide RNA sequence rather than reconstructing entire protein domains
2Ease of manufacture
If RNA-guided nucleases are used for genome editing, then cost-effectiveness and ease of generating target-specific editors is improved, but precision and efficiency in base editing and homology-directed repair remains insufficient
Solution Approach 1:
The patent segments the genome editing function into distinct modular components: the Cas nuclease module for DNA cleavage, guide RNA for target recognition, and separate donor DNA templates for precise repair. This segmentation allows optimization of each component independently, improving overall precision while maintaining cost-effectiveness
Solution Approach 2:
The invention introduces donor DNA templates as intermediary molecules that mediate the homology-directed repair process. These templates provide the precise sequence information needed for accurate editing, acting as a bridge between the nuclease-induced break and the desired final sequence
3Productivity
If double-stranded breaks are introduced via non-homologous end-joining for genome editing, then editing capability is achieved, but precision and control over the editing outcome is reduced due to error-prone repair
Solution Approach 1:
The patent employs a feedback mechanism where the guide RNA sequence is designed to match the desired edit outcome, and donor DNA templates provide feedback information for correct repair. This allows the system to self-correct and achieve precise editing outcomes by feedback-driven homology-directed repair rather than relying on error-prone non-homologous end-joining
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
Enhances the precision and efficiency of genome editing by enabling targeted sequence binding, cleavage, and modification, including non-homologous end-joining, homology-directed repair, and base editing, with improved cost-effectiveness and adaptability to eukaryotic cells.
Implementation Method 1
when bound to a guide RNA (gRNA) capable of hybridizing to the target DNA sequence
Implementation Method 2
The RGN polypeptide is capable of cleaving the target DNA sequence upon binding. The RGN polypeptide is capable of generating a double-stranded break. The RGN polypeptide is capable of generating a single-stranded break.
Implementation Method 3
Such RNA-guided nucleases can be used to edit genomes optionally through the introduction of a sequence-specific, double-stranded break that is repaired via error-prone non-homologous end-joining (NHEJ) to introduce a mutation at a specific genomic location.
Implementation Method 4
Alternatively, heterologous DNA may be introduced into the genomic site via homology-directed repair.
Implementation Method 5
RNA-guided nucleases (RGNs) can also be used for base editing when fused with a deaminase.
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.