Cell-Penetrating Peptide CRISPR RNP Composition for Low Off-Target Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing CRISPR-Cas-based gene correction technologies face challenges with low intracellular delivery efficiency, stability issues, and off-target effects, limiting their effectiveness for clinical and cell therapy applications.
Innovation Solution
A cell-penetrating peptide represented by General Formula 1, Arg-Arg-Arg-Trp-Cys-Lys-Arg-Arg-Arg-Ala-Ser-[Gly]m-[His-Glu]n, is used to enhance the delivery of a Cas protein-RNA complex (ribonucleoprotein: RNP) into cells, improving intracellular delivery efficiency and reducing off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Cas9 plasmid is delivered into cells, then gene correction function is provided, but intracellular delivery efficiency is low (around 10%)
Solution Approach 1:
A cell-penetrating peptide is introduced as an intermediary component that mediates the entry of Cas9 protein-RNA complex into cells. The peptide acts as a delivery vehicle that facilitates cellular uptake, thereby improving intracellular delivery efficiency from around 10% to significantly higher levels while maintaining the gene correction function of the Cas9 complex.
2Productivity
If Cas9 is expressed continuously in cells, then gene correction capability is maintained, but off-target effects increase
Solution Approach 1:
The system transitions from continuous Cas9 expression to transient, controlled delivery of Cas9 protein-RNA complex. The cell-penetrating peptide enables temporary presence of the editing machinery in cells, allowing gene correction to occur during a specific window while minimizing prolonged exposure that would cause off-target effects. This periodic action pattern reduces harmful off-target cleavage while maintaining necessary gene correction capability.
3Object-generated harmful factors
If protein-based RNP complex is used for delivery, then off-target effect is reduced, but intracellular delivery efficiency remains limited
Solution Approach 1:
The delivery system is constructed as a composite material combining Cas9 protein, guide RNA, and cell-penetrating peptide into a ribonucleoprotein complex. This composite structure integrates the gene editing functionality of Cas9-RNA with the cellular uptake capability of the peptide, thereby maintaining reduced off-target effects while significantly improving intracellular delivery efficiency beyond what the protein-RNA complex alone could achieve.
Data Source
AI summary
The present disclosure relates to a composition for enhancing the cell permeability and gene correction efficiency of Cas protein and guide RNA. The currently used CRISPR-Cas-based gene correction technology has the problems of difficult intracellular injection in a complex form, unverified stability and low efficiency even after injection, and the off-target problem. In contrast, the composition for gene correction of the present disclosure can be usefully used for gene therapy due to remarkably high intracellular delivery efficiency, inhibited off-target, and ensured stability.