A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable

Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address th...

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Published inPloS one Vol. 14; no. 1; p. e0210243
Main Authors Leynaud-Kieffer, Laure M. C., Curran, Samuel C., Kim, Irene, Magnuson, Jon K., Gladden, John M., Baker, Scott E., Simmons, Blake A.
Format Journal Article
LanguageEnglish
Published United States Public Library of Science 17.01.2019
Public Library of Science (PLoS)
Subjects
Aspergillus
Aspergillus niger
Aspergillus niger - genetics
BASIC BIOLOGICAL SCIENCES
Biodiesel fuels
Biology and Life Sciences
Biotechnology
CRISPR
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
DNA
Efficiency
Engineering and Technology
Enzymes
Fungi
Gene Editing - methods
Gene expression
Gene Targeting
Genetic aspects
Genetic engineering
Genetic modification
Genome editing
Genome, Fungal - genetics
Genomes
Genomics
Laboratories
Loci
Metabolic pathways
Mutation
Optimization
Proteins
Recombination
Research and Analysis Methods
Secretion
United States > US
California
Pacific Northwest
Online AccessGet full text

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Abstract Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "pop-out" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.
AbstractList Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A . niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific “pop-out” recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A . niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A . niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.
Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "pop-out" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "pop-out" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.
Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "pop-out" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.
Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "popout" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.
Audience Academic
Author Baker, Scott E.
Leynaud-Kieffer, Laure M. C.
Simmons, Blake A.
Curran, Samuel C.
Gladden, John M.
Kim, Irene
Magnuson, Jon K.
AuthorAffiliation 6 Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland, WA, United States of America
1 Swiss Federal Institute of Technology Lausanne, Lausanne, Vaud, Switzerland
Woosuk University, REPUBLIC OF KOREA
2 Joint Bioenergy Institute, Emeryville, CA, United States of America
3 Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States of America
8 Biosystems Design and Simulation Group, Environmental Molecular Sciences Division, Pacific Northwest National Laboratory, Richland, WA, United States of America
7 Department of Biomass Science and Conversion Technology, Sandia National Laboratories, Livermore, CA, United States of America
4 Comparative Biochemistry Graduate Group, University of California Berkeley, Berkeley, CA, United States of America
5 Department of Chemistry, University of California, Berkeley, CA, United States of America
AuthorAffiliation_xml – name: 8 Biosystems Design and Simulation Group, Environmental Molecular Sciences Division, Pacific Northwest National Laboratory, Richland, WA, United States of America
– name: 3 Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States of America
– name: Woosuk University, REPUBLIC OF KOREA
– name: 6 Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland, WA, United States of America
– name: 4 Comparative Biochemistry Graduate Group, University of California Berkeley, Berkeley, CA, United States of America
– name: 5 Department of Chemistry, University of California, Berkeley, CA, United States of America
– name: 2 Joint Bioenergy Institute, Emeryville, CA, United States of America
– name: 1 Swiss Federal Institute of Technology Lausanne, Lausanne, Vaud, Switzerland
– name: 7 Department of Biomass Science and Conversion Technology, Sandia National Laboratories, Livermore, CA, United States of America
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  surname: Leynaud-Kieffer
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  givenname: Samuel C.
  surname: Curran
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  surname: Kim
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BackLink https://www.ncbi.nlm.nih.gov/pubmed/30653574$$D View this record in MEDLINE/PubMed
https://www.osti.gov/servlets/purl/1508517$$D View this record in Osti.gov
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2019 Leynaud-Kieffer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.
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CorporateAuthor Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
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AC05-76RL01830; AC02-05CH11231
PNNL-SA-142504
USDOE Office of Science (SC), Biological and Environmental Research (BER)
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation and Fuels. Bioenergy Technologies Office (BETO)
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Vehicle Technologies Office (EE-3V)
Competing Interests: The authors have declared that no competing interests exist.
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Snippet Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while...
Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while...
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SubjectTerms Aspergillus
Aspergillus niger
Aspergillus niger - genetics
BASIC BIOLOGICAL SCIENCES
Biodiesel fuels
Biology and Life Sciences
Biotechnology
CRISPR
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
DNA
Efficiency
Engineering and Technology
Enzymes
Fungi
Gene Editing - methods
Gene expression
Gene Targeting
Genetic aspects
Genetic engineering
Genetic modification
Genome editing
Genome, Fungal - genetics
Genomes
Genomics
Laboratories
Loci
Metabolic pathways
Mutation
Optimization
Proteins
Recombination
Research and Analysis Methods
Secretion
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Title A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable
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