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A Versatile in Vivo DNA Assembly Toolbox for Fungal Strain Engineering
[Image: see text] Efficient homologous recombination in baker’s yeast allows accurate fusion of DNA fragments via short identical sequence tags in vivo. Eliminating the need for an Escherichia coli cloning step speeds up genetic engineering of this yeast and sets the stage for large high-throughput...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9594312/ https://www.ncbi.nlm.nih.gov/pubmed/36126183 http://dx.doi.org/10.1021/acssynbio.2c00159 |
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author | Jarczynska, Zofia Dorota Garcia Vanegas, Katherina Deichmann, Marcus Nørskov Jensen, Christina Scheeper, Marouschka Jasmijn Futyma, Malgorzata Ewa Strucko, Tomas Jares Contesini, Fabiano Sparholt Jørgensen, Tue Blæsbjerg Hoof, Jakob Hasbro Mortensen, Uffe |
author_facet | Jarczynska, Zofia Dorota Garcia Vanegas, Katherina Deichmann, Marcus Nørskov Jensen, Christina Scheeper, Marouschka Jasmijn Futyma, Malgorzata Ewa Strucko, Tomas Jares Contesini, Fabiano Sparholt Jørgensen, Tue Blæsbjerg Hoof, Jakob Hasbro Mortensen, Uffe |
author_sort | Jarczynska, Zofia Dorota |
collection | PubMed |
description | [Image: see text] Efficient homologous recombination in baker’s yeast allows accurate fusion of DNA fragments via short identical sequence tags in vivo. Eliminating the need for an Escherichia coli cloning step speeds up genetic engineering of this yeast and sets the stage for large high-throughput projects depending on DNA construction. With the aim of developing similar tools for filamentous fungi, we first set out to determine the genetic- and sequence-length requirements needed for efficient fusion reactions, and demonstrated that in nonhomologous end-joining deficient strains of Aspergillus nidulans, efficient fusions can be achieved by 25 bp sequence overlaps. Based on these results, we developed a novel fungal in vivo DNA assembly toolbox for simple and flexible genetic engineering of filamentous fungi. Specifically, we have used this method for construction of AMA1-based vectors, complex gene-targeting substrates for gene deletion and gene insertion, and for marker-free CRISPR based gene editing. All reactions were done via single-step transformations involving fusions of up to six different DNA fragments. Moreover, we show that it can be applied in four different species of Aspergilli. We therefore envision that in vivo DNA assembly can be advantageously used for many more purposes and will develop into a popular tool for fungal genetic engineering. |
format | Online Article Text |
id | pubmed-9594312 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-95943122022-10-26 A Versatile in Vivo DNA Assembly Toolbox for Fungal Strain Engineering Jarczynska, Zofia Dorota Garcia Vanegas, Katherina Deichmann, Marcus Nørskov Jensen, Christina Scheeper, Marouschka Jasmijn Futyma, Malgorzata Ewa Strucko, Tomas Jares Contesini, Fabiano Sparholt Jørgensen, Tue Blæsbjerg Hoof, Jakob Hasbro Mortensen, Uffe ACS Synth Biol [Image: see text] Efficient homologous recombination in baker’s yeast allows accurate fusion of DNA fragments via short identical sequence tags in vivo. Eliminating the need for an Escherichia coli cloning step speeds up genetic engineering of this yeast and sets the stage for large high-throughput projects depending on DNA construction. With the aim of developing similar tools for filamentous fungi, we first set out to determine the genetic- and sequence-length requirements needed for efficient fusion reactions, and demonstrated that in nonhomologous end-joining deficient strains of Aspergillus nidulans, efficient fusions can be achieved by 25 bp sequence overlaps. Based on these results, we developed a novel fungal in vivo DNA assembly toolbox for simple and flexible genetic engineering of filamentous fungi. Specifically, we have used this method for construction of AMA1-based vectors, complex gene-targeting substrates for gene deletion and gene insertion, and for marker-free CRISPR based gene editing. All reactions were done via single-step transformations involving fusions of up to six different DNA fragments. Moreover, we show that it can be applied in four different species of Aspergilli. We therefore envision that in vivo DNA assembly can be advantageously used for many more purposes and will develop into a popular tool for fungal genetic engineering. American Chemical Society 2022-09-20 2022-10-21 /pmc/articles/PMC9594312/ /pubmed/36126183 http://dx.doi.org/10.1021/acssynbio.2c00159 Text en © 2022 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Jarczynska, Zofia Dorota Garcia Vanegas, Katherina Deichmann, Marcus Nørskov Jensen, Christina Scheeper, Marouschka Jasmijn Futyma, Malgorzata Ewa Strucko, Tomas Jares Contesini, Fabiano Sparholt Jørgensen, Tue Blæsbjerg Hoof, Jakob Hasbro Mortensen, Uffe A Versatile in Vivo DNA Assembly Toolbox for Fungal Strain Engineering |
title | A Versatile in
Vivo DNA Assembly Toolbox for Fungal
Strain Engineering |
title_full | A Versatile in
Vivo DNA Assembly Toolbox for Fungal
Strain Engineering |
title_fullStr | A Versatile in
Vivo DNA Assembly Toolbox for Fungal
Strain Engineering |
title_full_unstemmed | A Versatile in
Vivo DNA Assembly Toolbox for Fungal
Strain Engineering |
title_short | A Versatile in
Vivo DNA Assembly Toolbox for Fungal
Strain Engineering |
title_sort | versatile in
vivo dna assembly toolbox for fungal
strain engineering |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9594312/ https://www.ncbi.nlm.nih.gov/pubmed/36126183 http://dx.doi.org/10.1021/acssynbio.2c00159 |
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