Functional Characterization of Novel U6 RNA Polymerase III Promoters: Their Implication for CRISPR-Cas9-Mediated Gene Editing in Aspergillus oryzae

Curr Microbiol. 2019 Dec;76(12):1443-1451. doi: 10.1007/s00284-019-01770-0. Epub 2019 Sep 20.

Abstract

U6 RNA polymerase III promoter (PU6), which is a key element in controlling the generation of single-guide RNA (sgRNA) for gene editing through CRISPR-Cas9 system, was investigated in this work. Using bioinformatics approach, two novel U6 ribonucleic acid (U6 RNA) sequences of Aspergillus niger were identified, showing that they had conserved motifs similar to other U6 RNAs. The putative PU6 located at the upstream sequence of A. niger U6 RNA exhibited the consensus motif, CCAATYA, and the TATA box which shared highly conserved characteristics across Aspergilli, whereas the A- and B-boxes were found at the intragenic and downstream of the structural genes, respectively. Using Aspergillus oryzae as a workhorse system, the function of A. niger PU6s for controlling the transcripts of sgRNA was verified, in which the orotidine-5'-phosphate decarboxylase (pyrG) sequence was used as a target for gene disruption through CRISPR-Cas9 system. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis of the selected pyrG auxotrophic strains showed the expression of sgRNA, indicating that the non-native promoters could efficiently drive sgRNA expression in A. oryzae. These identified promoters are useful genetic tools for precise engineering of metabolic pathways in the industrially important fungus through the empowered CRISPR-Cas9-associated gene-editing system.

MeSH terms

  • Aspergillus oryzae / enzymology
  • Aspergillus oryzae / genetics*
  • CRISPR-Cas Systems*
  • Fungal Proteins / genetics*
  • Fungal Proteins / metabolism
  • Gene Editing*
  • Gene Expression Regulation, Fungal
  • Promoter Regions, Genetic*
  • RNA Polymerase III / genetics*
  • RNA Polymerase III / metabolism
  • RNA, Fungal / genetics*
  • RNA, Fungal / metabolism
  • RNA, Guide / genetics*
  • RNA, Guide / metabolism

Substances

  • Fungal Proteins
  • RNA, Fungal
  • RNA, Guide
  • RNA Polymerase III