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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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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: Oct 31, 2025

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
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Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli.

Enrico König1, Francesca Zerbini1, Ilaria Zanella1

  • 1Synthetic and Structural Vaccinology Unit, CIBIO, University of Trento, Via Sommarive, 9, Trento, Italy.

Bio-Protocol
|June 28, 2021
PubMed
Summary

This study introduces a new CRISPR/Cas9 gene editing protocol for Escherichia coli. The method enables efficient, stepwise gene knockouts in E. coli, facilitating high-throughput research and industrial applications.

Keywords:
BiotechnologyDouble-stranded donor DNAGenome editingHigh-throughputSynthetic biology

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Last Updated: Oct 31, 2025

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Area of Science:

  • Molecular Biology
  • Genomics
  • Microbiology

Background:

  • CRISPR/Cas9 technology is a revolutionary genome editing tool.
  • Escherichia coli is a vital bacterium for research and industry.

Purpose of the Study:

  • To develop a simple, robust, and effective CRISPR/Cas9 protocol for gene knockout in E. coli.
  • To enable efficient, stepwise gene knockouts for high-throughput applications.

Main Methods:

  • Utilized CRISPR/Cas9 system combined with λ Red machinery.
  • Employed double-stranded donor DNA and a plasmid curing strategy.
  • Developed a protocol for gene knockout in E. coli.

Main Results:

  • Achieved high mutagenesis efficiencies for gene knockouts.
  • Enabled multiple, stepwise gene knockouts.
  • Protocol allows for a new mutation after two working days.

Conclusions:

  • The presented protocol is a significant advancement for genome editing in E. coli.
  • This method is applicable for both research and industrial high-throughput approaches.
  • Simplifies and enhances gene editing capabilities in E. coli.