In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | https://hdl.handle.net/1822/77903 |
Resumo: | Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems. |
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In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA ComplexesCRISPRCas9gene knockoutCL-DNAlipoplexplasmidgene deliverymultivalent cationic lipidsMVL5Science & TechnologyClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems.This research was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and the Project FCOMP-01– 0124-FEDER-021053 (PTDC/SAU-BMA/121028/2010). This research was also supported by the Microfluidic Layer-by-layer Assembly of Cationic Liposome—Nucleic Acid Nanoparticles for Gene Delivery project (032520) co-funded by FCT and the ERDF through COMPETE2020. Diana A. Sousa (D.A.S) and Celso J.O. Ferreira (C.J.O.F) acknowledge FCT for the grants PD/BD/139083/2018 and SFRH/BD/149199/2019, respectively.info:eu-repo/semantics/publishedVersionMultidisciplinary Digital Publishing Institute (MDPI)Universidade do MinhoSousa, Diana AndradeGaspar, RicardoFerreira, Celso J. O.Baltazar, FátimaRodrigues, L. R.Silva, Bruno F. B.2022-05-192022-05-19T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/1822/77903engSousa, Diana; Gaspar, Ricardo; Ferreira, Celso J. O.; Baltazar, Fátima; Rodrigues, Lígia R.; Silva, Bruno F. B., In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes. Pharmaceutics, 14(5), 1087, 20221999-492310.3390/pharmaceutics14051087https://www.mdpi.com/1999-4923/14/5/1087info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T12:25:02Zoai:repositorium.sdum.uminho.pt:1822/77903Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:19:13.290437Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes |
title |
In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes |
spellingShingle |
In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes Sousa, Diana Andrade CRISPR Cas9 gene knockout CL-DNA lipoplex plasmid gene delivery multivalent cationic lipids MVL5 Science & Technology |
title_short |
In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes |
title_full |
In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes |
title_fullStr |
In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes |
title_full_unstemmed |
In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes |
title_sort |
In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes |
author |
Sousa, Diana Andrade |
author_facet |
Sousa, Diana Andrade Gaspar, Ricardo Ferreira, Celso J. O. Baltazar, Fátima Rodrigues, L. R. Silva, Bruno F. B. |
author_role |
author |
author2 |
Gaspar, Ricardo Ferreira, Celso J. O. Baltazar, Fátima Rodrigues, L. R. Silva, Bruno F. B. |
author2_role |
author author author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Sousa, Diana Andrade Gaspar, Ricardo Ferreira, Celso J. O. Baltazar, Fátima Rodrigues, L. R. Silva, Bruno F. B. |
dc.subject.por.fl_str_mv |
CRISPR Cas9 gene knockout CL-DNA lipoplex plasmid gene delivery multivalent cationic lipids MVL5 Science & Technology |
topic |
CRISPR Cas9 gene knockout CL-DNA lipoplex plasmid gene delivery multivalent cationic lipids MVL5 Science & Technology |
description |
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-05-19 2022-05-19T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://hdl.handle.net/1822/77903 |
url |
https://hdl.handle.net/1822/77903 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Sousa, Diana; Gaspar, Ricardo; Ferreira, Celso J. O.; Baltazar, Fátima; Rodrigues, Lígia R.; Silva, Bruno F. B., In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes. Pharmaceutics, 14(5), 1087, 2022 1999-4923 10.3390/pharmaceutics14051087 https://www.mdpi.com/1999-4923/14/5/1087 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Multidisciplinary Digital Publishing Institute (MDPI) |
publisher.none.fl_str_mv |
Multidisciplinary Digital Publishing Institute (MDPI) |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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