Development of a CRISPR/Cas9-based tool for gene deletion in Issatchenkia orientalis

Vinh G. Tran, Mingfeng Cao, Zia Fatma, Xiaofei Song, Huimin Zhao

Research output: Contribution to journalArticle

Abstract

The nonconventional yeast Issatchenkia orientalis has emerged as a potential platform microorganism for production of organic acids due to its ability to grow robustly under highly acidic conditions. However, lack of efficient genetic tools remains a major bottleneck in metabolic engineering of this organism. Here we report that the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) was functional for plasmid replication in I. orientalis, and the resulting episomal plasmid enabled efficient genome editing by the CRISPR/Cas9 system. The optimized CRISPR/Cas9-based system employed a fusion RPR1'-tRNA promoter for single guide RNA (sgRNA) expression and could attain greater than 97% gene disruption efficiency for various gene targets. Additionally, we demonstrated multiplexed gene deletion with disruption efficiencies of 90% and 47% for double gene and triple gene knockouts, respectively. This genome editing tool can be used for rapid strain development and metabolic engineering of this organism for production of biofuels and chemicals.

Original languageEnglish (US)
Article numbere00345-19
JournalmSphere
Volume4
Issue number3
DOIs
StatePublished - May 1 2019

Keywords

  • CRISPR/Cas9
  • Genome editing
  • Issatchenkia orientalis
  • Metabolic engineering
  • Synthetic biology

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Fingerprint Dive into the research topics of 'Development of a CRISPR/Cas9-based tool for gene deletion in Issatchenkia orientalis'. Together they form a unique fingerprint.

  • Cite this