Marker-free genome editing in Ustilago trichophora with the CRISPR-Cas9 technology

RNA Biol. 2019 Apr;16(4):397-403. doi: 10.1080/15476286.2018.1493329. Epub 2018 Aug 10.

Abstract

In this communication, we report the adaptation of the CRISPR-Cas9 technology in Ustilago trichophora prototrophic wild-type isolate obtained from its natural host Echinochloa crus-galli. The established CRISPR vector and method enable a rapid and marker-free introduction of Cas9-induced non-homologous end-joining (NHEJ) dependent mutation at the targeted gene. Moreover, the method allows a specific modification of the chromosomal DNA sequence by Cas9-induced homologous recombination using short DNA repair templates. The results demonstrate the applicability of the CRISPR-Cas9 technology in U. trichophora for both gene knock-out by the NHEJ pathway and specific gene modification by templated genome editing, paving the way for rapid metabolic engineering of this Ustilago species for industrial applications.

Keywords: CRISPR; Cas9; Ustilago trichophora; genome editing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems / genetics*
  • Chromosomes, Fungal / genetics
  • Gene Editing*
  • Genetic Markers
  • Genome, Fungal
  • Homologous Recombination / genetics
  • Ustilago / genetics*

Substances

  • Genetic Markers
  • CRISPR-Associated Protein 9

Grants and funding

The results described were generated in one of the projects of the ‘Innovationsallianz ZeroCarbFP’ that was supported by the Bundesministerium für Bildung und Forschung within the ‘Innovationsinitiative industrielle Biotechnologie’ (FKZ 031B0181F);Bundesministerium für Bildung und Forschung [FKZ 031B0181F].