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dMAD7 is a promising tool for targeted gene regulation in the methylotrophic yeast Komagataella phaffii.
Krappinger, Julian C; Aguilar Gomez, Carla M; Hoenikl, Andrea; Schusterbauer, Veronika; Hatzl, Anna-Maria; Feichtinger, Julia; Glieder, Anton.
Afiliación
  • Krappinger JC; Christian Doppler Laboratory for Innovative Pichia pastoris Host and Vector Systems, Graz, Austria; Division of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.
  • Aguilar Gomez CM; Christian Doppler Laboratory for Innovative Pichia pastoris Host and Vector Systems, Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria.
  • Hoenikl A; Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria.
  • Schusterbauer V; bisy GmbH, Hofstaetten, Austria.
  • Hatzl AM; Christian Doppler Laboratory for Innovative Pichia pastoris Host and Vector Systems, Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria.
  • Feichtinger J; Christian Doppler Laboratory for Innovative Pichia pastoris Host and Vector Systems, Graz, Austria; Division of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria. Electronic address: julia.feichtinger@medunigraz.at.
  • Glieder A; Christian Doppler Laboratory for Innovative Pichia pastoris Host and Vector Systems, Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria.
N Biotechnol ; 83: 110-120, 2024 Nov 25.
Article en En | MEDLINE | ID: mdl-38960022
ABSTRACT
The methylotrophic yeast Komagataella phaffii is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant "dead" MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research. CRISPRi utilizing dMAD7 does not introduce double-strand breaks but only binds to the DNA to regulate gene expression. Here, we report the first use of dMAD7 in K. phaffii to regulate the expression of the enhanced green fluorescent protein (eGFP). A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. Integration loci/events of investigated strains were assessed through whole genome sequencing. Additionally, RNA-sequencing experiments corroborated the whole genome sequencing results and showed a significantly reduced expression of eGFP in strains containing a dMAD7 plasmid, among others. Our findings conclusively demonstrate the utility of dMAD7 in K. phaffii through successfully regulating eGFP expression.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Fluorescentes Verdes / Saccharomycetales Idioma: En Revista: N Biotechnol Asunto de la revista: BIOLOGIA MOLECULAR / ENGENHARIA BIOMEDICA Año: 2024 Tipo del documento: Article País de afiliación: Austria Pais de publicación: Países Bajos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Fluorescentes Verdes / Saccharomycetales Idioma: En Revista: N Biotechnol Asunto de la revista: BIOLOGIA MOLECULAR / ENGENHARIA BIOMEDICA Año: 2024 Tipo del documento: Article País de afiliación: Austria Pais de publicación: Países Bajos