High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells
Genome editing based on dual CRISPR-Cas9 complexes (multiplexes) permits removing specific genomic sequences in living cells leveraging research on functional genomics and genetic therapies. Delivering the required large and multicomponent reagents in a synchronous and stoichiometric manner remains,...
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| Format: | Article |
| Language: | English |
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Elsevier
2023-03-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253123000458 |
| _version_ | 1827994144057851904 |
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| author | Francesca Tasca Marcella Brescia Jin Liu Josephine M. Janssen Kamel Mamchaoui Manuel A.F.V. Gonçalves |
| author_facet | Francesca Tasca Marcella Brescia Jin Liu Josephine M. Janssen Kamel Mamchaoui Manuel A.F.V. Gonçalves |
| author_sort | Francesca Tasca |
| collection | DOAJ |
| description | Genome editing based on dual CRISPR-Cas9 complexes (multiplexes) permits removing specific genomic sequences in living cells leveraging research on functional genomics and genetic therapies. Delivering the required large and multicomponent reagents in a synchronous and stoichiometric manner remains, however, challenging. Moreover, uncoordinated activity of independently acting CRISPR-Cas9 multiplexes increases the complexity of genome editing outcomes. Here, we investigate the potential of fostering precise multiplexing genome editing using high-capacity adenovector particles (AdVPs) for the delivery of Cas9 ortholog fusion constructs alone (forced Cas9 heterodimers) or together with their cognate guide RNAs (forced CRISPR-Cas9 heterodimers). We demonstrate that the efficiency and accuracy of targeted chromosomal DNA deletions achieved by single AdVPs encoding forced CRISPR-Cas9 heterodimers is superior to that obtained when the various components are delivered separately. Finally, all-in-one AdVP delivery of forced CRISPR-Cas9 heterodimers triggers robust DMD exon 51 splice site excision resulting in reading frame restoration and selection-free detection of dystrophin in muscle cells derived from Duchenne muscular dystrophy patients. In conclusion, AdVPs promote precise multiplexing genome editing through the integrated delivery of forced CRISPR-Cas9 heterodimer components, which, in comparison with split conventional CRISPR-Cas9 multiplexes, engage target sequences in a more coordinated fashion. |
| first_indexed | 2024-04-10T04:35:28Z |
| format | Article |
| id | doaj.art-bd14d663ff8f4e04a1281686f5c0d2d9 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-04-10T04:35:28Z |
| publishDate | 2023-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-bd14d663ff8f4e04a1281686f5c0d2d92023-03-10T04:34:51ZengElsevierMolecular Therapy: Nucleic Acids2162-25312023-03-0131746762High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cellsFrancesca Tasca0Marcella Brescia1Jin Liu2Josephine M. Janssen3Kamel Mamchaoui4Manuel A.F.V. Gonçalves5Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the NetherlandsDepartment of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the NetherlandsDepartment of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the NetherlandsDepartment of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the NetherlandsSorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, Paris, FranceDepartment of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands; Corresponding author: Manuel A.F.V. Gonçalves, Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.Genome editing based on dual CRISPR-Cas9 complexes (multiplexes) permits removing specific genomic sequences in living cells leveraging research on functional genomics and genetic therapies. Delivering the required large and multicomponent reagents in a synchronous and stoichiometric manner remains, however, challenging. Moreover, uncoordinated activity of independently acting CRISPR-Cas9 multiplexes increases the complexity of genome editing outcomes. Here, we investigate the potential of fostering precise multiplexing genome editing using high-capacity adenovector particles (AdVPs) for the delivery of Cas9 ortholog fusion constructs alone (forced Cas9 heterodimers) or together with their cognate guide RNAs (forced CRISPR-Cas9 heterodimers). We demonstrate that the efficiency and accuracy of targeted chromosomal DNA deletions achieved by single AdVPs encoding forced CRISPR-Cas9 heterodimers is superior to that obtained when the various components are delivered separately. Finally, all-in-one AdVP delivery of forced CRISPR-Cas9 heterodimers triggers robust DMD exon 51 splice site excision resulting in reading frame restoration and selection-free detection of dystrophin in muscle cells derived from Duchenne muscular dystrophy patients. In conclusion, AdVPs promote precise multiplexing genome editing through the integrated delivery of forced CRISPR-Cas9 heterodimer components, which, in comparison with split conventional CRISPR-Cas9 multiplexes, engage target sequences in a more coordinated fashion.http://www.sciencedirect.com/science/article/pii/S2162253123000458MT: RNA/DNA Editingmultiplexing gene editinggene deliveryfusion Cas9 proteinshigh-capacity adenoviral vectorson-target specificity |
| spellingShingle | Francesca Tasca Marcella Brescia Jin Liu Josephine M. Janssen Kamel Mamchaoui Manuel A.F.V. Gonçalves High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells Molecular Therapy: Nucleic Acids MT: RNA/DNA Editing multiplexing gene editing gene delivery fusion Cas9 proteins high-capacity adenoviral vectors on-target specificity |
| title | High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells |
| title_full | High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells |
| title_fullStr | High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells |
| title_full_unstemmed | High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells |
| title_short | High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells |
| title_sort | high capacity adenovector delivery of forced crispr cas9 heterodimers fosters precise chromosomal deletions in human cells |
| topic | MT: RNA/DNA Editing multiplexing gene editing gene delivery fusion Cas9 proteins high-capacity adenoviral vectors on-target specificity |
| url | http://www.sciencedirect.com/science/article/pii/S2162253123000458 |
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