Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei

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Abstract

Trichoderma reesei is a filamentous fungus that is used worldwide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement using mutagenesis and screening approaches. It is also desirable to specifically manipulate the genes of the organism to further improve and to modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation, we have developed a simple transient method for silencing genes in T. reesei. Using gene-specific small interfering RNAs (siRNAs) targeted to mus53, we could achieve up to 90% knockdown of mus53 mRNA. As a practical example, we demonstrated that transient silencing of DNA repair genes significantly improved homologous integration of DNA at a specific locus in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59% marker gene integration.

Original languageEnglish
Article numbere00535-17
Number of pages11
JournalApplied and Environmental Microbiology
Volume83
Issue number15
DOIs
Publication statusPublished - 2017
MoE publication typeA1 Journal article-refereed

Fingerprint

Trichoderma reesei
Trichoderma
DNA repair
DNA Repair
repair
Fungi
fungus
small interfering RNA
DNA
protoplasts
fungi
Protoplasts
gene
Small Interfering RNA
Genes
genes
homologous recombination
gene silencing
RNA
mutagenesis

Keywords

  • Trichoderma reesei
  • filamentous fungi
  • gene silencing
  • industrial biotechnology
  • siRNA

Cite this

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title = "Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei",
abstract = "Trichoderma reesei is a filamentous fungus that is used worldwide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement using mutagenesis and screening approaches. It is also desirable to specifically manipulate the genes of the organism to further improve and to modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation, we have developed a simple transient method for silencing genes in T. reesei. Using gene-specific small interfering RNAs (siRNAs) targeted to mus53, we could achieve up to 90{\%} knockdown of mus53 mRNA. As a practical example, we demonstrated that transient silencing of DNA repair genes significantly improved homologous integration of DNA at a specific locus in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59{\%} marker gene integration.",
keywords = "Trichoderma reesei, filamentous fungi, gene silencing, industrial biotechnology, siRNA",
author = "Chum, {Pak Yang} and Georg Schmidt and Markku Saloheimo and Landowski, {Christopher Paul}",
year = "2017",
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T1 - Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei

AU - Chum, Pak Yang

AU - Schmidt, Georg

AU - Saloheimo, Markku

AU - Landowski, Christopher Paul

PY - 2017

Y1 - 2017

N2 - Trichoderma reesei is a filamentous fungus that is used worldwide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement using mutagenesis and screening approaches. It is also desirable to specifically manipulate the genes of the organism to further improve and to modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation, we have developed a simple transient method for silencing genes in T. reesei. Using gene-specific small interfering RNAs (siRNAs) targeted to mus53, we could achieve up to 90% knockdown of mus53 mRNA. As a practical example, we demonstrated that transient silencing of DNA repair genes significantly improved homologous integration of DNA at a specific locus in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59% marker gene integration.

AB - Trichoderma reesei is a filamentous fungus that is used worldwide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement using mutagenesis and screening approaches. It is also desirable to specifically manipulate the genes of the organism to further improve and to modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation, we have developed a simple transient method for silencing genes in T. reesei. Using gene-specific small interfering RNAs (siRNAs) targeted to mus53, we could achieve up to 90% knockdown of mus53 mRNA. As a practical example, we demonstrated that transient silencing of DNA repair genes significantly improved homologous integration of DNA at a specific locus in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59% marker gene integration.

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