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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Related Experiment Video

Updated: Sep 24, 2025

Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira
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CRISPR/Cas9-Based Methods for Inactivating Actinobacterial Biosynthetic Genes and Elucidating Function.

Audam Chhun1, Fabrizio Alberti2

  • 1Biophore - DMF, University of Lausanne, Lausanne, Switzerland.

Methods in Molecular Biology (Clifton, N.J.)
|May 6, 2022
PubMed
Summary

This study presents a CRISPR/Cas9 genome engineering protocol for Streptomyces, enabling rapid gene deletion. Metabolite analysis then identifies the function of deleted genes in secondary metabolism.

Keywords:
ActinobacteriaBiosynthetic geneCRISPR/Cas9Natural productspCRISPomyces-2

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • Actinobacteria, particularly Streptomyces, are prolific producers of secondary metabolites with significant pharmaceutical applications.
  • Understanding the genetic basis of secondary metabolism is crucial for unlocking novel bioactive compounds.
  • Efficient genome engineering tools are needed to study gene function in these complex organisms.

Purpose of the Study:

  • To establish a standardized CRISPR/Cas9 protocol for marker-less gene deletion in Streptomyces species.
  • To demonstrate the utility of this method for investigating genes involved in secondary metabolism.
  • To correlate gene inactivation with changes in metabolite production.

Main Methods:

  • Utilized the pCRISPomyces-2 CRISPR/Cas9 vector for targeted gene deletion.
  • Developed a protocol for efficient genome engineering in Streptomyces.
  • Performed comparative metabolite analysis of wild-type and gene-deleted strains.

Main Results:

  • Successfully deleted a specific biosynthetic gene in a Streptomyces species using the CRISPR/Cas9 system.
  • Metabolite profiling revealed significant alterations in the secondary metabolome following gene inactivation.
  • The observed metabolic changes allowed for the putative assignment of the deleted gene's function.

Conclusions:

  • The described CRISPR/Cas9 protocol provides a fast and efficient method for genome engineering in Streptomyces.
  • This approach facilitates the study of secondary metabolism by enabling targeted gene function analysis.
  • The integration of metabolite analysis is key to elucidating gene roles in biosynthetic pathways.