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CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis
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Visualized Multigene Editing System for Aspergillus niger.

Cen Li1,2,3, Jingwen Zhou1,2,3,4, Shengqi Rao5

  • 1National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

ACS Synthetic Biology
|September 24, 2021
PubMed
Summary
This summary is machine-generated.

Researchers developed a visualized multigene editing system (VMS) for Aspergillus niger. This system improves the screening of gene-edited colonies, enabling more efficient genetic modification in this fungus.

Keywords:
Aspergillus nigerCRISPR/Cas9endogenous tRNAmultigene editingvisualized pigment gene

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Area of Science:

  • Molecular Biology
  • Mycology
  • Genetic Engineering

Background:

  • CRISPR/Cas9 system delivery into Aspergillus niger can be hindered by resistance markers, complicating gene editing.
  • Efficient screening of transformed colonies is crucial for successful genetic manipulation in Aspergillus niger.
  • Targeting multiple genes simultaneously presents challenges in strain development and selection.

Purpose of the Study:

  • To design and implement a visualized multigene editing system (VMS) for Aspergillus niger.
  • To enhance the efficiency of screening positive colonies after transformation.
  • To facilitate the development of homokaryotic multigene editing strains.

Main Methods:

  • A novel visualized multigene editing system (VMS) was designed using a unique tRNA-guide RNA (gRNA) array.
  • The array incorporated gRNAs for a pigment gene (albA) and multiple target genes (ammA, amyA, prtT, kusA, glaA).
  • Disruption of albA resulted in white colonies, serving as a visual marker for successful transformation and editing.

Main Results:

  • The VMS demonstrated effective screening of edited colonies, with disruption efficiencies for one to five target genes reaching up to 89.2%.
  • Specific tRNA sequences (tRNAAla, tRNA Phe, tRNA Arg, tRNA Ile, and tRNA Leu) were utilized to enhance gRNA release.
  • The disruption rates decreased with an increasing number of target genes, indicating a dose-dependent effect.

Conclusions:

  • The developed VMS provides an effective and visually guided method for screening multigene edited strains of Aspergillus niger.
  • This system significantly improves the efficiency of identifying homokaryotic edited strains, overcoming previous screening limitations.
  • The VMS is a valuable tool for accelerating genetic engineering and functional genomics studies in Aspergillus niger.