CRISPR Delivery Nanoparticle Complex with Polycation Escape Polymer
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Solution Overview
Problem
Current methods for delivering CRISPR-Cas9 systems into cells face challenges in efficient endosomal escape and targeted gene modification, leading to low efficacy and specificity.
Innovation Solution
A complex comprising a nanoparticle-nucleic acid conjugate, a Type II or Type V CRISPR system with a site-directed DNA-modifying polypeptide and guide RNA, and a polycation-based endosomal escape polymer is developed, allowing for encapsulation and targeted delivery of the CRISPR system into cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-Cas9 systems are delivered into cells using current methods, then gene modification can be achieved, but endosomal escape efficiency is low and delivery efficacy is poor
Solution Approach 1:
The patent employs a composite delivery system consisting of a cationic polymer (for endosomal escape), a nanoparticle (for targeted delivery and protection), and the CRISPR-Cas9 complex. This multi-component composite structure addresses the limitations of single-method delivery by combining the advantages of each component: the cationic polymer facilitates endosomal escape through membrane disruption, the nanoparticle provides targeted delivery and protects the cargo, and together they achieve high delivery efficacy and reliable gene modification.
2Manufacturing precision
If current delivery methods are used, then some gene modification occurs, but specificity is low and off-target effects increase
Solution Approach 1:
The patent enhances delivery specificity through localized functionalization of the nanoparticle surface with targeting ligands that recognize specific cell surface markers. This local quality modification ensures that the CRISPR-Cas9 complex is delivered only to the intended target cells, improving gene modification specificity and reducing off-target effects in non-target tissues.
3Stability of the object's composition
If encapsulation is used to protect the CRISPR system, then delivery stability improves, but release efficiency from endosomes may be reduced
Solution Approach 1:
The patent utilizes parameter changes in the endosomal environment (pH gradient from pH 6.5 in endosomes to pH 7.4 in cytoplasm) to trigger the conformational change of the cationic polymer. This parameter-based triggering mechanism allows the polymer to maintain a compact, protective conformation during circulation (stability) and automatically switch to an expanded, membrane-disrupting conformation in the acidic endosomal environment (release efficiency), resolving the contradiction between protection and release.
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 complex achieves high specificity and low toxicity, with a significant percentage of cells remaining viable and exhibiting effective gene modification, demonstrating improved delivery and efficacy compared to existing methods.
Implementation Method 1
a polycation-based endosomal escape polymer
Implementation Method 2
endosomal escape polymer
Implementation Method 3
a guide RNA... for target recognition
Implementation Method 4
Cas9 protein functions as an RNA-guided endonuclease that... generate double-stranded DNA breaks
Implementation Method 5
a nanoparticle-nucleic acid conjugate... allowing for encapsulation and targeted delivery
Data Source
Figure 1A
Figure 1B
Figure 2A~2B
AI summary
The present disclosure provides a complex comprising a nanoparticle; a Type II or a Type V CRISPR system comprising a site -directed DNA-modifying polypeptide and a guide RNA; and a polycation-based endosomal escape polymer. The present disclosure provides methods of making and using a complex of the present disclosure.