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Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction.
- Source :
-
Pharmaceutics . Oct2023, Vol. 15 Issue 10, p2500. 20p. - Publication Year :
- 2023
-
Abstract
- The therapeutic potential of the CRISPR-Cas9 gene editing system in treating numerous genetic disorders is immense. To fully realize this potential, it is crucial to achieve safe and efficient delivery of CRISPR-Cas9 components into the nuclei of target cells. In this study, we investigated the applicability of the amphipathic cell-penetrating peptide LAH5, previously employed for DNA delivery, in the intracellular delivery of spCas9:sgRNA ribonucleoprotein (RNP) and the RNP/single-stranded homology-directed repair (HDR) template. Our findings reveal that the LAH5 peptide effectively formed nanocomplexes with both RNP and RNP/HDR cargo, and these nanocomplexes demonstrated successful cellular uptake and cargo delivery. The loading of all RNP/HDR components into LAH5 nanocomplexes was confirmed using an electrophoretic mobility shift assay. Functional screening of various ratios of peptide/RNP nanocomplexes was performed on fluorescent reporter cell lines to assess gene editing and HDR-mediated gene correction. Moreover, targeted gene editing of the CCR5 gene was successfully demonstrated across diverse cell lines. This LAH5-based delivery strategy represents a significant advancement toward the development of therapeutic delivery systems for CRISPR-Cas-based genetic engineering in in vitro and ex vivo applications. [ABSTRACT FROM AUTHOR]
- Subjects :
- *GENOME editing
*GENETIC engineering
*PEPTIDES
*CRISPRS
*CELL nuclei
*CELL lines
Subjects
Details
- Language :
- English
- ISSN :
- 19994923
- Volume :
- 15
- Issue :
- 10
- Database :
- Academic Search Index
- Journal :
- Pharmaceutics
- Publication Type :
- Academic Journal
- Accession number :
- 173319798
- Full Text :
- https://doi.org/10.3390/pharmaceutics15102500