Fine-tuning the regulation of Cas9 expression levels for efficient CRISPR-Cas9 mediated recombination in Streptomyces

Suhui Ye, Behnam Enghiad, Huimin Zhao, Eriko Takano

Research output: Contribution to journalArticlepeer-review

Abstract

CRISPR-Cas9 has proven as a very powerful gene editing tool for Actinomyces, allowing scarless and precise genome editing in selected strains of these biotechnologically relevant microorganisms. However, its general application in actinomycetes has been limited due to its inefficacy when applying the system in an untested strain. Here, we provide evidence of how Cas9 levels are toxic for the model actinomycetes Streptomyces coelicolor M145 and Streptomyces lividans TK24, which show delayed or absence of growth. We overcame this toxicity by lowering Cas9 levels and have generated a set of plasmids in which Cas9 expression is either controlled by theophylline-inducible or constitutive promoters. We validated the targeting of these CRISPR-Cas9 system using the glycerol uptake operon and the actinorhodin biosynthesis gene cluster. Our results highlight the importance of adjusting Cas9 expression levels specifically in strains to gain optimum and efficient gene editing in Actinomyces.

Original languageEnglish (US)
Pages (from-to)413-423
Number of pages11
JournalJournal of Industrial Microbiology and Biotechnology
Volume47
Issue number4-5
DOIs
StatePublished - May 1 2020

Keywords

  • CRISPR-Cas9
  • Genome editing
  • Glycerol
  • Streptomyces
  • Theophylline riboswitch

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Fingerprint Dive into the research topics of 'Fine-tuning the regulation of Cas9 expression levels for efficient CRISPR-Cas9 mediated recombination in Streptomyces'. Together they form a unique fingerprint.

Cite this