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Related Concept Videos

CRISPR and crRNAs02:53

CRISPR and crRNAs

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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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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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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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Protocol for NT-CRISPR: A Method for Efficient Genome Engineering in Vibrio natriegens.

Daniel Stukenberg1,2, Josef Hoff1,2,3, Anna Faber1,2,4

  • 1Center for Synthetic Microbiology, Philipps-Universität Marburg, Marburg, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|October 3, 2024
PubMed
Summary

We present NT-CRISPR, a novel genome engineering method for the fast-growing bacterium Vibrio natriegens. This tool facilitates genetic modifications, enabling diverse research and biotechnological applications.

Keywords:
BiotechnologyCRISPR-Cas9ChassisGenome engineeringGolden Gate cloningNatural transformationSynthetic biologyVibrio natriegens

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Vibrio natriegens is a gram-negative bacterium noted for its exceptionally rapid growth rate (doubling time < 10 minutes).
  • The fast growth of V. natriegens makes it a promising candidate for various research and biotechnological applications.
  • Efficient genome engineering tools are crucial for harnessing the potential of V. natriegens.

Purpose of the Study:

  • To introduce NT-CRISPR, a new method for genome engineering in Vibrio natriegens.
  • To provide a detailed protocol for utilizing the NT-CRISPR tool.
  • To enable a broader range of basic research and biotechnological applications using V. natriegens.

Main Methods:

  • NT-CRISPR combines natural transformation with CRISPR-Cas9 counterselection.
  • This method allows for precise genetic modifications within the V. natriegens genome.
  • The protocol details the steps for implementing this genome engineering technique.

Main Results:

  • The NT-CRISPR system enables the deletion of specific genomic regions.
  • Foreign DNA sequences can be successfully integrated into the V. natriegens genome.
  • Point mutations can be introduced, and up to three simultaneous genetic modifications are achievable.

Conclusions:

  • NT-CRISPR is an effective tool for genome engineering in Vibrio natriegens.
  • The method supports versatile genetic modifications, including deletions, integrations, and point mutations.
  • This tool expands the possibilities for research and biotechnology applications utilizing fast-growing V. natriegens.