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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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CRISPR01:59

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this...
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Updated: Aug 11, 2025

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria.

Daphne Collias1,2, Elena Vialetto1, Jiaqi Yu1

  • 1Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), 97080, Würzburg, Germany.

Nature Communications
|February 8, 2023
PubMed
Summary
This summary is machine-generated.

Researchers developed a new CRISPR-Cas genome editing method for bacteria. By reducing DNA targeting, this approach enhances RecA-mediated repair, enabling efficient editing even in strains with low transformation efficiencies.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Bacterial genome editing often uses Cas nucleases for counter-selection.
  • Efficient editing typically requires high transformation efficiencies and recombination, which are limited in many bacterial strains.

Purpose of the Study:

  • To demonstrate that attenuating DNA targeting activity can enable RecA-mediated repair for bacterial genome editing.
  • To explore methods for achieving CRISPR-driven editing in bacteria with low transformation efficiencies.

Main Methods:

  • Systematically attenuating DNA targeting activity of CRISPR-Cas systems.
  • Modifying guide RNA (gRNA) format or expression, using nucleases with reduced cleavage activity, or engineering attenuated gRNAs (atgRNAs).
  • Applying atgRNAs for genome editing in Escherichia coli, Klebsiella oxytoca, and Klebsiella pneumoniae.

Main Results:

  • Attenuating DNA targeting significantly increased cell counts and improved editing efficiency for Cas9 and Cas12a.
  • Engineered atgRNAs with features like disruptive hairpins or guide mismatches enhanced editing.
  • Successfully restored ampicillin sensitivity in Klebsiella pneumoniae using atgRNAs, creating a resistance marker.

Conclusions:

  • Systematically attenuating DNA targeting activity is a viable strategy to drive CRISPR-Cas genome editing in diverse bacteria.
  • This approach overcomes limitations of low transformation efficiencies and enables RecA-mediated repair for genome modification.
  • The development of attenuated gRNAs offers a versatile tool for bacterial genetic studies and engineering.