Öktem, M.; Mastrobattista, E.; de Jong, O.G. Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction. Pharmaceutics 2023, 15, 2500. https://doi.org/10.3390/pharmaceutics15102500
Öktem, M.; Mastrobattista, E.; de Jong, O.G. Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction. Pharmaceutics 2023, 15, 2500. https://doi.org/10.3390/pharmaceutics15102500
Öktem, M.; Mastrobattista, E.; de Jong, O.G. Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction. Pharmaceutics 2023, 15, 2500. https://doi.org/10.3390/pharmaceutics15102500
Öktem, M.; Mastrobattista, E.; de Jong, O.G. Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction. Pharmaceutics 2023, 15, 2500. https://doi.org/10.3390/pharmaceutics15102500
Abstract
The CRISPR/Cas9 gene editing system has enormous potential in the treatment of multiple genetic disorders. To fully harness this potential, it is essential to deliver CRISPR-Cas9 components safely and effectively into the nuclei of target cells. In this study, we tested the amphipathic cell-penetrating peptide LAH5, previously used for DNA delivery, to intracellularly deliver spCas9: sgRNA ribonucleoprotein (RNP) and RNP/single-stranded homology-directed repair (HDR) template. We discovered that the LAH5 peptide effectively formed nanocomplexes with both RNP and RNP/HDR cargo, and that these nanocomplexes were taken up by cells effectively. Peptide/RNP nanocomplexes were characterized for size and charge, and nanocomplexation with RNP and RNP/HDR cargo was demonstrated via an electrophoretic mobility shift assay. Various ratios of peptide/RNP nanocomplexes were functionally screened on fluorescent reporter cell lines for gene editing and HDR-mediated gene correction. Additionally, targeted gene editing of the CCR5 gene was demonstrated in various cell lines. This LAH5-based delivery strategy paves the way for the development of therapeutic delivery systems for CRISPR/Cas-based genomic engineering.
Medicine and Pharmacology, Medicine and Pharmacology
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