Modified Nuclease-gRNA Compositions for Precise Eukaryotic Editing
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Solution Overview
Problem
CRISPR/Cas systems face limitations in eukaryotic organisms, including low editing efficiency, off-target events, and target sequence preferences, as well as challenges in delivery and expression of the nuclease.
Innovation Solution
Compositions comprising nucleases with specific amino acid sequences and guide RNAs (gRNAs) with high identity to SEQ ID NOs, optionally with nuclear localization sequences (NLS) and stem-loop structures, are used for targeted nucleic acid modification, including vectors with promoters for efficient delivery and expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If CRISPR/Cas systems are used for nucleic acid editing, then versatility and ease of use are improved, but editing efficiency and off-target effects worsen
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of guide RNAs through the incorporation of modified nucleotides (such as 2'-O-methyl modifications, phosphorothioate linkages, and locked nucleic acids). These structural parameter changes enhance the stability, binding affinity, and specificity of the guide RNA, thereby improving editing efficiency while maintaining ease of use. The modified nucleotides alter physical-chemical parameters like thermal stability and resistance to nucleases, directly addressing the reliability issue.
2Ease of operation
If CRISPR/Cas systems are used for nucleic acid editing, then versatility and ease of use are improved, but off-target events worsen
Solution Approach 1:
The patent employs parameter changes by introducing modified nucleotides with altered base-pairing properties and enhanced specificity. Modifications such as 2'-O-methyl groups and locked nucleic acid structures change the thermodynamic parameters of RNA-DNA hybridization, allowing for more precise discrimination between target and off-target sequences. This improves specificity without compromising the ease of use of the CRISPR/Cas system.
3Reliability
If nucleases are delivered to eukaryotic cells, then gene editing capability is improved, but delivery efficiency and expression worsen
Solution Approach 1:
The patent uses intermediaries in the form of modified nucleotides and nucleic acid structures that facilitate cellular uptake and nuclear delivery. The modified guide RNAs with enhanced stability and binding properties act as intermediaries that improve the overall delivery efficiency. Additionally, the use of chemically modified oligonucleotides serves as intermediary molecules that protect against degradation during delivery and enhance cellular uptake, thereby improving productivity without compromising gene editing capability.
Data Source
AI summary
The present disclosure provides nucleases and compositions, methods, and systems thereof for nucleic acid modification. More particularly, the present disclosure provides compositions and system comprising a nuclease comprising an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1-250 and at least one gRNA for target nucleic acid modification.