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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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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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Homologous Recombination02:31

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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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Conservative Site-specific Recombination and Phase Variation02:53

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Updated: Jan 5, 2026

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH.

Youxiang Liang1, Song Jiao1, Miaomiao Wang1

  • 1Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China; Key Laboratory of Industrial Biocatalysis, Tsinghua University, Ministry of Education, Beijing, 100084, China.

Metabolic Engineering
|October 15, 2019
PubMed
Summary

We developed a CRISPR/Cas9 genome editing system for Rhodococcus ruber, enhancing its efficiency for bio-production applications. This system enables precise genetic modifications, improving acrylamide production and biocatalyst performance.

Keywords:
Acrylamide bio-productionCRISPR/Cas9Genome editingRecombinaseRestriction-modification systemRhodococcus

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Area of Science:

  • Microbiology
  • Biotechnology
  • Synthetic Biology

Background:

  • Rhodococcus species are valuable for bioconversion, biosynthesis, and bioremediation due to their adaptability and metabolic diversity.
  • Efficient genome editing in Rhodococcus is hindered by high GC-content and low transformation/recombination efficiencies.

Purpose of the Study:

  • To establish a robust CRISPR/Cas9-based genome editing system for Rhodococcus ruber.
  • To improve transformation and editing efficiencies for genetic manipulation.
  • To engineer R. ruber for enhanced bio-production of acrylamide.

Main Methods:

  • Developed a CRISPR/Cas9 system for R. ruber, bypassing the restriction-modification system to increase transformation efficiency 89-fold.
  • Incorporated bacteriophage recombinases (Che9c60 and Che9c61) to boost editing efficiency from 1% to 75%.
  • Established a triple-plasmid recombineering system for efficient gene deletion, insertion, and mutation.

Main Results:

  • Achieved a significant increase in transformation efficiency, facilitating mutant screening.
  • Demonstrated high editing efficiency (75%) using the developed system.
  • Engineered R. ruber THY for acrylamide bio-production, increasing yield to 500 µg/L and reducing byproducts to 0.5 µg/L.
  • Enhanced biocatalyst recyclability from 1 to 4 batches.

Conclusions:

  • The developed CRISPR/Cas9 system overcomes previous limitations in Rhodococcus genome editing.
  • Engineered R. ruber THY shows improved performance for industrial acrylamide production.
  • This platform is valuable for metabolic engineering of Rhodococcus species.