Self-assembled Ribonucleoprotein Nanoparticles for CRISPR Delivery
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Solution Overview
Problem
Current methods for delivering the CRISPR/Cas9 system for gene modification, such as viral vectors and electroporation, raise safety concerns and have limited efficiency due to integration issues and cell damage, while non-viral vehicles like cationic lipids and cell-penetrating peptides still require improvement in delivery efficiency.
Innovation Solution
A polyribonucleotide-protein complex comprising a polyribonucleotide with multiple repeating units of single guide RNA (sgRNA) and small interfering RNA (siRNA) regions, combined with nuclease proteins like Cas9, forms nanoparticles that can self-assemble for enhanced intracellular delivery and target gene disruption.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If viral vectors or electroporation are used to deliver CRISPR/Cas9 system, then delivery efficiency is improved, but safety concerns arise due to integration issues and cell damage
Solution Approach 1:
The patent extracts the CRISPR/Cas9 system from plasmid form and delivers it directly as a ribonucleoprotein complex, eliminating the need for viral vectors or electroporation. This removes the harmful integration and cell damage effects while maintaining delivery efficiency through direct RNP complex introduction into cells
Solution Approach 2:
The patent uses a peptide-RNA conjugate as an intermediary carrier to deliver the CRISPR/Cas9 RNP complex. This conjugate acts as a safe mediator that facilitates cellular uptake without causing the harmful effects associated with viral vectors or electroporation, achieving both efficient delivery and cell safety
2Object-affected harmful factors
If non-viral vehicles like cationic lipids and cell-penetrating peptides are used to deliver CRISPR/Cas9, then safety is improved, but delivery efficiency remains limited
Solution Approach 1:
The patent merges the advantages of cell-penetrating peptides with CRISPR/Cas9 RNP complex delivery. The peptide-RNA conjugate combines the cell-penetration capability with the CRISPR system, achieving both high delivery efficiency and cell safety without the limitations of using either component separately
Solution Approach 2:
The patent creates a composite delivery system consisting of peptide-RNA conjugates that combine the properties of cell-penetrating peptides with guide RNA. This composite material achieves superior delivery efficiency compared to conventional non-viral vehicles while maintaining cell safety
3Adaptability or versatility
If plasmids encoding Cas9 and sgRNA are transfected into target cells, then gene modification capability is achieved, but safety concerns arise from vector DNA integration
Solution Approach 1:
The patent extracts the gene modification capability from plasmid-based systems and implements it through direct RNP complex delivery. This eliminates the plasmid DNA that could integrate into the genome and cause genetic malfunctions, while preserving the ability to modify target genes through the delivered Cas9-sgRNA complex
Solution Approach 2:
The patent performs preliminary assembly of the CRISPR/Cas9 system as a pre-formed ribonucleoprotein complex with all necessary components (Cas9 protein, guide RNA, and peptide carrier) assembled before delivery. This preliminary action ensures that the system functions immediately upon cellular entry without requiring plasmid transfection and expression, eliminating integration risks
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
The polyribonucleotide-protein complex effectively suppresses target gene expression in eukaryotic cells with improved stability and delivery efficiency, reducing cytotoxicity and increasing gene disruption rates compared to traditional methods, as demonstrated by cellular and in vivo experiments.
Implementation Method 1
forms nanoparticles that can self-assemble for enhanced intracellular delivery
Data Source
AI summary
Provided are a self-assembled polyribonucleotide-protein complex including a polyribonucleotide including a plurality of first repeating units having a single guide RNA (sgRNA) region and a small interfering RNA (siRNA) region; and one or more nuclease proteins binding to the sgRNA region, and use thereof.