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CRISPR/dCas9-Mediated Multiplex Gene Repression in Streptomyces.

Yawei Zhao1,2, Lei Li3, Guosong Zheng3

  • 1State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.

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|June 5, 2018
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Summary
This summary is machine-generated.

Researchers developed a new CRISPR interference (CRISPRi) system for multiplex gene repression in Streptomyces coelicolor. This tool enables simultaneous repression of multiple genes, aiding in functional screening and metabolic engineering of these important bacteria.

Keywords:
CRISPRiStreptomycesdCas9multiplex gene repression

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Streptomyces bacteria produce valuable secondary metabolites but are genetically challenging to manipulate.
  • Existing CRISPR/Cas9 tools facilitate genome editing, but CRISPR interference (CRISPRi) was limited to single-gene repression.
  • Efficient genetic tools are crucial for understanding and engineering Streptomyces for drug discovery and industrial applications.

Purpose of the Study:

  • To develop a novel CRISPR interference (CRISPRi) system for simultaneous repression of multiple genes in Streptomyces coelicolor.
  • To demonstrate the system's efficacy for functional gene screening and identify novel genes involved in bacterial growth.
  • To provide a versatile tool for genetic manipulation in Streptomyces, applicable to other strains for metabolic engineering.

Main Methods:

  • Development of an integrative CRISPRi system using the pSET152 plasmid backbone in Streptomyces coelicolor.
  • Constitutive promoters were employed for the expression of the dCas9 and single-guide RNA (sgRNA) complex.
  • Validation of multiplex gene repression by quantifying mRNA levels of four target genes.

Main Results:

  • The novel integrative CRISPRi system achieved efficient simultaneous repression of multiple genes.
  • Target gene mRNA levels were reduced to 2-32% of control levels, demonstrating significant interference.
  • The system was successfully used for functional gene screening, identifying an orphan response regulator (SCO2013) involved in bacterial growth.

Conclusions:

  • The developed integrative CRISPRi system is highly effective for multiplex gene repression in Streptomyces coelicolor.
  • This tool significantly advances genetic manipulation capabilities in Streptomyces, facilitating functional gene screening.
  • The system holds potential for broader application in metabolic engineering and research across various Streptomyces species.