Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction

Mert Öktem; Enrico Mastrobattista; Olivier G de Jong

doi:10.3390/pharmaceutics15102500

Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction

Mert Öktem
, Enrico Mastrobattista
, Olivier G de Jong^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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.

Original language	English
Article number	2500
Pages (from-to)	1-20
Number of pages	20
Journal	Pharmaceutics
Volume	15
Issue number	10
DOIs	https://doi.org/10.3390/pharmaceutics15102500
Publication status	Published - 20 Oct 2023

Bibliographical note

Publisher Copyright:
© 2023 by the authors.

Funding

This research was funded by the Türkiye Ministry of National Education. M.Ö. is supported by the PhD scholarship provided by the Republic of Türkiye Ministry of National Education. O.G.d.J. is supported by a Veni grant from the Science Domain of the Dutch Research Council (NWO) (grant number VI.Veni.192.174). The authors thank Dario Fenenko and Thai Hoang Nguyen for their help with the optimization of the electrophoretic mobility shift assay.

Funders	Funder number
Science Domain of the Dutch Research Council
Türkiye Ministry of National Education
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	VI.Veni.192.174

Keywords

CRISPR-Cas9
HDR
LAH5
RNP
cell-penetrating peptide (CPP)
delivery

Access to Document

10.3390/pharmaceutics15102500Licence: CC BY

pharmaceutics-15-02500Final published version, 4.13 MBLicence: CC BY

Cite this

@article{ab47ace1cd614b16b8f436e0fc58afa3,

title = "Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction",

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. ",

keywords = "CRISPR-Cas9, HDR, LAH5, RNP, cell-penetrating peptide (CPP), delivery",

author = "Mert {\"O}ktem and Enrico Mastrobattista and \{de Jong\}, \{Olivier G\}",

note = "Publisher Copyright: {\textcopyright} 2023 by the authors.",

year = "2023",

month = oct,

day = "20",

doi = "10.3390/pharmaceutics15102500",

language = "English",

volume = "15",

pages = "1--20",

journal = "Pharmaceutics",

issn = "1999-4923",

publisher = "American Chemical Society",

number = "10",

}

TY - JOUR

T1 - Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction

AU - Öktem, Mert

AU - Mastrobattista, Enrico

AU - de Jong, Olivier G

PY - 2023/10/20

Y1 - 2023/10/20

N2 - 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.

AB - 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.

KW - CRISPR-Cas9

KW - HDR

KW - LAH5

KW - RNP

KW - cell-penetrating peptide (CPP)

KW - delivery

UR - https://www.scopus.com/pages/publications/85175089403

U2 - 10.3390/pharmaceutics15102500

DO - 10.3390/pharmaceutics15102500

M3 - Article

C2 - 37896260

SN - 1999-4923

VL - 15

SP - 1

EP - 20

JO - Pharmaceutics

JF - Pharmaceutics

IS - 10

M1 - 2500

ER -

Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction

Abstract

Bibliographical note

Funding

Keywords

Access to Document

Other files and links

Fingerprint

Cite this