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Genetic barcodes allow traceability of CRISPR/Cas9-derived Aspergillus niger strains without affecting their fitness

Safe use of genetically modified organisms (GMOs) in biotechnology requires the ability to track the presence of these strains in any environment in which they are applied. For this, introduction of genetic barcodes within the editing site represents a valuable tool for the identification of microbi...

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Autores principales: Garrigues, Sandra, Kun, Roland S., de Vries, Ronald P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254718/
https://www.ncbi.nlm.nih.gov/pubmed/33723654
http://dx.doi.org/10.1007/s00294-021-01164-5
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author Garrigues, Sandra
Kun, Roland S.
de Vries, Ronald P.
author_facet Garrigues, Sandra
Kun, Roland S.
de Vries, Ronald P.
author_sort Garrigues, Sandra
collection PubMed
description Safe use of genetically modified organisms (GMOs) in biotechnology requires the ability to track the presence of these strains in any environment in which they are applied. For this, introduction of genetic barcodes within the editing site represents a valuable tool for the identification of microbial strains that have undergone genetic modifications. However, it is not known whether these barcodes would have any unexpected effect in the resulting strains or affect the efficiency of the genetic modification. CRISPR/Cas9 has become one of the fastest-growing technologies for genome editing in a range of organisms, including fungi. However, this technology enables the generation of scarless GMOs that are very difficult to distinguish from naturally occurring mutants or other modified organisms. In this study, we address this issue using the industrial workhorse Aspergillus niger as a test case. We applied CRISPR/Cas9 technology to delete the genes encoding the transcriptional regulators XlnR and AraR, involved in the production of plant biomass-degrading enzymes. We generated 20-bp barcoded and non-barcoded ΔxlnR and ΔaraR mutants and analyzed the traceability and fitness of the resulting strains, as well as the efficiency of the genetic modification. Results showed that both barcoded and non-barcoded mutants can be traced by routine PCR reactions when the specific CRISPR/Cas9 modification is known. Additionally, barcodes neither affected the efficiency of the genetic modification nor the growth or protein production of the resulting strains. These results confirm the suitability of genetic barcodes to trace CRISPR-derived GMOs without affecting the performance of the resulting strains. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00294-021-01164-5.
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spelling pubmed-82547182021-07-20 Genetic barcodes allow traceability of CRISPR/Cas9-derived Aspergillus niger strains without affecting their fitness Garrigues, Sandra Kun, Roland S. de Vries, Ronald P. Curr Genet Technical Note Safe use of genetically modified organisms (GMOs) in biotechnology requires the ability to track the presence of these strains in any environment in which they are applied. For this, introduction of genetic barcodes within the editing site represents a valuable tool for the identification of microbial strains that have undergone genetic modifications. However, it is not known whether these barcodes would have any unexpected effect in the resulting strains or affect the efficiency of the genetic modification. CRISPR/Cas9 has become one of the fastest-growing technologies for genome editing in a range of organisms, including fungi. However, this technology enables the generation of scarless GMOs that are very difficult to distinguish from naturally occurring mutants or other modified organisms. In this study, we address this issue using the industrial workhorse Aspergillus niger as a test case. We applied CRISPR/Cas9 technology to delete the genes encoding the transcriptional regulators XlnR and AraR, involved in the production of plant biomass-degrading enzymes. We generated 20-bp barcoded and non-barcoded ΔxlnR and ΔaraR mutants and analyzed the traceability and fitness of the resulting strains, as well as the efficiency of the genetic modification. Results showed that both barcoded and non-barcoded mutants can be traced by routine PCR reactions when the specific CRISPR/Cas9 modification is known. Additionally, barcodes neither affected the efficiency of the genetic modification nor the growth or protein production of the resulting strains. These results confirm the suitability of genetic barcodes to trace CRISPR-derived GMOs without affecting the performance of the resulting strains. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00294-021-01164-5. Springer Berlin Heidelberg 2021-03-16 2021 /pmc/articles/PMC8254718/ /pubmed/33723654 http://dx.doi.org/10.1007/s00294-021-01164-5 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Technical Note
Garrigues, Sandra
Kun, Roland S.
de Vries, Ronald P.
Genetic barcodes allow traceability of CRISPR/Cas9-derived Aspergillus niger strains without affecting their fitness
title Genetic barcodes allow traceability of CRISPR/Cas9-derived Aspergillus niger strains without affecting their fitness
title_full Genetic barcodes allow traceability of CRISPR/Cas9-derived Aspergillus niger strains without affecting their fitness
title_fullStr Genetic barcodes allow traceability of CRISPR/Cas9-derived Aspergillus niger strains without affecting their fitness
title_full_unstemmed Genetic barcodes allow traceability of CRISPR/Cas9-derived Aspergillus niger strains without affecting their fitness
title_short Genetic barcodes allow traceability of CRISPR/Cas9-derived Aspergillus niger strains without affecting their fitness
title_sort genetic barcodes allow traceability of crispr/cas9-derived aspergillus niger strains without affecting their fitness
topic Technical Note
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254718/
https://www.ncbi.nlm.nih.gov/pubmed/33723654
http://dx.doi.org/10.1007/s00294-021-01164-5
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