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Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus
CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterolog...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9303279/ https://www.ncbi.nlm.nih.gov/pubmed/35801912 http://dx.doi.org/10.1093/nar/gkac587 |
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author | Aulicino, Francesco Pelosse, Martin Toelzer, Christine Capin, Julien Ilegems, Erwin Meysami, Parisa Rollarson, Ruth Berggren, Per-Olof Dillingham, Mark Simon Schaffitzel, Christiane Saleem, Moin A Welsh, Gavin I Berger, Imre |
author_facet | Aulicino, Francesco Pelosse, Martin Toelzer, Christine Capin, Julien Ilegems, Erwin Meysami, Parisa Rollarson, Ruth Berggren, Per-Olof Dillingham, Mark Simon Schaffitzel, Christiane Saleem, Moin A Welsh, Gavin I Berger, Imre |
author_sort | Aulicino, Francesco |
collection | PubMed |
description | CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity. |
format | Online Article Text |
id | pubmed-9303279 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-93032792022-07-22 Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus Aulicino, Francesco Pelosse, Martin Toelzer, Christine Capin, Julien Ilegems, Erwin Meysami, Parisa Rollarson, Ruth Berggren, Per-Olof Dillingham, Mark Simon Schaffitzel, Christiane Saleem, Moin A Welsh, Gavin I Berger, Imre Nucleic Acids Res Synthetic Biology and Bioengineering CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity. Oxford University Press 2022-07-08 /pmc/articles/PMC9303279/ /pubmed/35801912 http://dx.doi.org/10.1093/nar/gkac587 Text en © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Synthetic Biology and Bioengineering Aulicino, Francesco Pelosse, Martin Toelzer, Christine Capin, Julien Ilegems, Erwin Meysami, Parisa Rollarson, Ruth Berggren, Per-Olof Dillingham, Mark Simon Schaffitzel, Christiane Saleem, Moin A Welsh, Gavin I Berger, Imre Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus |
title | Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus |
title_full | Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus |
title_fullStr | Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus |
title_full_unstemmed | Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus |
title_short | Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus |
title_sort | highly efficient crispr-mediated large dna docking and multiplexed prime editing using a single baculovirus |
topic | Synthetic Biology and Bioengineering |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9303279/ https://www.ncbi.nlm.nih.gov/pubmed/35801912 http://dx.doi.org/10.1093/nar/gkac587 |
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