NmeCas9 Polynucleotides Codon Optimization Liver Editing
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Solution Overview
Problem
Existing approaches for expressing RNA-guided DNA binding agents like CRISPR-Cas systems in mammals, such as NmeCas9, often result in suboptimal expression levels and immunogenicity, leading to undesirable cytokine elevations, which limits their effectiveness in genome editing applications.
Innovation Solution
The development of polynucleotides encoding NmeCas9 with optimized coding sequences, codon usage, and additional features like nuclear localization signals and linker sequences, which enhance expression levels and reduce immunogenicity, specifically designed for improved editing efficiency in mammalian cells and organs like the liver.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing polynucleotides encoding NmeCas9 are used for genome editing in mammals, then the basic genome editing function is achieved, but expression levels are suboptimal and immunogenicity increases leading to undesirable cytokine elevations
Solution Approach 1:
The patent applies parameter changes by optimizing codon usage in the NmeCas9 coding sequence to match mammalian cellular preferences, modifying nucleotide sequences to reduce immunogenic recognition, and adjusting structural parameters by adding nuclear localization signals. These parameter modifications maintain the genome editing function while reducing immunogenicity and improving expression levels in mammalian cells.
Solution Approach 2:
The patent creates a composite polynucleotide structure that combines the NmeCas9 coding sequence with additional functional elements including nuclear localization signals (NLS), optimized codon usage patterns, and modified nucleotide sequences. This composite construction integrates multiple functional components into a single polynucleotide molecule that achieves both effective genome editing and reduced immunogenicity.
2Productivity
If existing polynucleotides encoding NmeCas9 are used, then the basic function is maintained, but expression levels remain suboptimal for effective genome editing
Solution Approach 1:
The patent optimizes expression levels by changing nucleotide sequence parameters including codon usage to match mammalian preferences, adding nuclear localization signals to enhance nuclear import efficiency, and modifying UTR regions to improve mRNA stability and translation efficiency. These parameter changes collectively enhance productivity while maintaining editing reliability.
3Productivity
If polynucleotides with optimized coding sequences and codon usage are designed, then expression levels and activity improve, but the complexity of polynucleotide design increases
Solution Approach 1:
The patent applies local quality by making targeted modifications to specific regions of the polynucleotide rather than redesigning the entire structure. Nuclear localization signals are added at specific locations (N-terminal or C-terminal), codon optimization is applied locally to the coding sequence, and UTR modifications are made at terminal regions. This localized approach enhances expression without requiring complete structural redesign.
Solution Approach 2:
The patent segments the polynucleotide into distinct functional modules including the coding sequence, nuclear localization signals, 5' UTR, and 3' UTR regions. Each segment is independently optimized for its specific function, allowing modular design that simplifies the overall complexity while achieving enhanced expression levels.
Data Source
AI summary
Compositions and methods for gene editing are provided. In some embodiments, provided is a polynucleotide encoding an RNA-guided DNA binding agent such as N. meningitidis Cas9 that can provide one or more of improved editing efficiency, reduced immunogenicity, or other benefits.