Development of microtiter plate scale CRISPR/Cas9 transformation method for Aspergillus niger based on in vitro assembled ribonucleoprotein complexes

Joosu Kuivanen (Corresponding Author), Veera Korja, Sami Holmström, Peter Richard

Research output: Contribution to journalArticleScientificpeer-review

Abstract

Background: The CRISPR/Cas9 is currently the predominant technology to enhance the genome editing efficiency in eukaryotes. Established tools for many fungal species exist while most of them are based on in vivo expressed Cas9 and guide RNA (gRNA). Alternatively, in vitro assembled Cas9 and gRNA ribonucleoprotein complexes can be used in genome editing, however, only a few examples have been reported in fungi. In general, high-throughput compatible transformation workflows for filamentous fungi are immature.

Results: In this study, a CRISPR/Cas9 facilitated transformation and genome editing method based on in vitro assembled ribonucleoprotein complexes was developed for the filamentous fungus Aspergillus niger. The method was downscaled to be compatible with 96-well microtiter plates. The optimized method resulted in 100% targeting efficiency for a single genomic target. After the optimization, the method was demonstrated to be suitable for multiplexed genome editing with two or three genomic targets in a metabolic engineering application. As a result, an A. niger strain with improved capacity to produce galactarate, a potential chemical building block, was generated.

Conclusions: The developed microtiter plate compatible CRISPR/Cas9 method provides a basis for high-throughput genome editing workflows in A. niger and other related species. In addition, it improves the cost-effectiveness of CRISPR/Cas9 genome editing methods in fungi based on in vitro assembled ribonucleoproteins. The demonstrated metabolic engineering example with multiplexed genome editing highlights the applicability of the method.

Original languageEnglish
Number of pages12
JournalFungal Biology and Biotechnology
Volume6
Issue number3
DOIs
Publication statusPublished - 15 Mar 2019
MoE publication typeNot Eligible

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Clustered Regularly Interspaced Short Palindromic Repeats
Ribonucleoproteins
Aspergillus niger
Fungi
Guide RNA
Metabolic Engineering
Workflow
Gene Editing
In Vitro Techniques
Eukaryota
Cost-Benefit Analysis
Technology

Keywords

  • CRISPR
  • genome editing
  • high-throughput
  • automation
  • aspergillus niger
  • galactarate
  • mucic acid
  • pectin
  • galacturonate
  • metabolic engineering

Cite this

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title = "Development of microtiter plate scale CRISPR/Cas9 transformation method for Aspergillus niger based on in vitro assembled ribonucleoprotein complexes",
abstract = "Background: The CRISPR/Cas9 is currently the predominant technology to enhance the genome editing efficiency in eukaryotes. Established tools for many fungal species exist while most of them are based on in vivo expressed Cas9 and guide RNA (gRNA). Alternatively, in vitro assembled Cas9 and gRNA ribonucleoprotein complexes can be used in genome editing, however, only a few examples have been reported in fungi. In general, high-throughput compatible transformation workflows for filamentous fungi are immature.Results: In this study, a CRISPR/Cas9 facilitated transformation and genome editing method based on in vitro assembled ribonucleoprotein complexes was developed for the filamentous fungus Aspergillus niger. The method was downscaled to be compatible with 96-well microtiter plates. The optimized method resulted in 100{\%} targeting efficiency for a single genomic target. After the optimization, the method was demonstrated to be suitable for multiplexed genome editing with two or three genomic targets in a metabolic engineering application. As a result, an A. niger strain with improved capacity to produce galactarate, a potential chemical building block, was generated. Conclusions: The developed microtiter plate compatible CRISPR/Cas9 method provides a basis for high-throughput genome editing workflows in A. niger and other related species. In addition, it improves the cost-effectiveness of CRISPR/Cas9 genome editing methods in fungi based on in vitro assembled ribonucleoproteins. The demonstrated metabolic engineering example with multiplexed genome editing highlights the applicability of the method.",
keywords = "CRISPR, genome editing, high-throughput, automation, aspergillus niger, galactarate, mucic acid, pectin, galacturonate, metabolic engineering",
author = "Joosu Kuivanen and Veera Korja and Sami Holmstr{\"o}m and Peter Richard",
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Development of microtiter plate scale CRISPR/Cas9 transformation method for Aspergillus niger based on in vitro assembled ribonucleoprotein complexes. / Kuivanen, Joosu (Corresponding Author); Korja, Veera; Holmström, Sami; Richard, Peter.

In: Fungal Biology and Biotechnology, Vol. 6, No. 3, 15.03.2019.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Development of microtiter plate scale CRISPR/Cas9 transformation method for Aspergillus niger based on in vitro assembled ribonucleoprotein complexes

AU - Kuivanen, Joosu

AU - Korja, Veera

AU - Holmström, Sami

AU - Richard, Peter

PY - 2019/3/15

Y1 - 2019/3/15

N2 - Background: The CRISPR/Cas9 is currently the predominant technology to enhance the genome editing efficiency in eukaryotes. Established tools for many fungal species exist while most of them are based on in vivo expressed Cas9 and guide RNA (gRNA). Alternatively, in vitro assembled Cas9 and gRNA ribonucleoprotein complexes can be used in genome editing, however, only a few examples have been reported in fungi. In general, high-throughput compatible transformation workflows for filamentous fungi are immature.Results: In this study, a CRISPR/Cas9 facilitated transformation and genome editing method based on in vitro assembled ribonucleoprotein complexes was developed for the filamentous fungus Aspergillus niger. The method was downscaled to be compatible with 96-well microtiter plates. The optimized method resulted in 100% targeting efficiency for a single genomic target. After the optimization, the method was demonstrated to be suitable for multiplexed genome editing with two or three genomic targets in a metabolic engineering application. As a result, an A. niger strain with improved capacity to produce galactarate, a potential chemical building block, was generated. Conclusions: The developed microtiter plate compatible CRISPR/Cas9 method provides a basis for high-throughput genome editing workflows in A. niger and other related species. In addition, it improves the cost-effectiveness of CRISPR/Cas9 genome editing methods in fungi based on in vitro assembled ribonucleoproteins. The demonstrated metabolic engineering example with multiplexed genome editing highlights the applicability of the method.

AB - Background: The CRISPR/Cas9 is currently the predominant technology to enhance the genome editing efficiency in eukaryotes. Established tools for many fungal species exist while most of them are based on in vivo expressed Cas9 and guide RNA (gRNA). Alternatively, in vitro assembled Cas9 and gRNA ribonucleoprotein complexes can be used in genome editing, however, only a few examples have been reported in fungi. In general, high-throughput compatible transformation workflows for filamentous fungi are immature.Results: In this study, a CRISPR/Cas9 facilitated transformation and genome editing method based on in vitro assembled ribonucleoprotein complexes was developed for the filamentous fungus Aspergillus niger. The method was downscaled to be compatible with 96-well microtiter plates. The optimized method resulted in 100% targeting efficiency for a single genomic target. After the optimization, the method was demonstrated to be suitable for multiplexed genome editing with two or three genomic targets in a metabolic engineering application. As a result, an A. niger strain with improved capacity to produce galactarate, a potential chemical building block, was generated. Conclusions: The developed microtiter plate compatible CRISPR/Cas9 method provides a basis for high-throughput genome editing workflows in A. niger and other related species. In addition, it improves the cost-effectiveness of CRISPR/Cas9 genome editing methods in fungi based on in vitro assembled ribonucleoproteins. The demonstrated metabolic engineering example with multiplexed genome editing highlights the applicability of the method.

KW - CRISPR

KW - genome editing

KW - high-throughput

KW - automation

KW - aspergillus niger

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KW - mucic acid

KW - pectin

KW - galacturonate

KW - metabolic engineering

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JO - Fungal Biology and Biotechnology

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SN - 2054-3085

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