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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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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

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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.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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Improving <i>Bacillus subtilis</i> as Biological Chassis Performance by the CRISPR Genetic Toolkit.

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High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots
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A Programmable CRISPR/Cas9 Toolkit Improves Lycopene Production in Bacillus subtilis.

Yang Liu1, Haijiao Cheng1, Haoni Li1,2

  • 1Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.

Applied and Environmental Microbiology
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This study introduces a CRISPR/Cas9 toolkit for efficient metabolic engineering of Bacillus subtilis, streamlining gene integration and expression for producing valuable compounds like lycopene.

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Bacillus subtilisCRISPRMEP pathwaygenome editinglycopenemicrobial fermentation

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Area of Science:

  • Synthetic Biology
  • Microbial Engineering
  • Biotechnology

Background:

  • Bacillus subtilis is a safe and widely used host for producing recombinant proteins and chemicals.
  • Metabolic engineering of B. subtilis is crucial but hindered by limited selective markers, especially for complex pathways.
  • Efficient genetic tools are needed to overcome these limitations in B. subtilis.

Purpose of the Study:

  • To develop an easy-to-use CRISPR/Cas9-based cloning toolkit for B. subtilis metabolic engineering.
  • To address challenges in chromosomal integration, promoter selection, terminator function, and guide RNA targeting.
  • To demonstrate the toolkit's utility in optimizing the production of industrially relevant compounds.

Main Methods:

  • Utilized CRISPR/Cas9 technology to construct a versatile cloning toolkit for B. subtilis.
  • Characterized six promoters (0.9- to 23-fold P43 strength) and seven terminators (>90% efficiency).
  • Designed six guide RNA targets and confirmed up to 100% integration efficiency using a GFP reporter.

Main Results:

  • Developed a toolkit enabling rapid cloning and one-step subcloning for stable chromosome integration in B. subtilis.
  • Demonstrated successful optimization of lycopene production by manipulating 13 key genes in the biosynthetic pathway.
  • Identified specific gene clusters (ispG-idi-dxs-ispD positive, dxr-ispE-ispF-ispH negative) impacting lycopene yield.

Conclusions:

  • The developed toolkit significantly facilitates pathway assembly and gene expression in B. subtilis.
  • This strategy enables rapid engineering of B. subtilis strains for complex chemical and pharmaceutical production.
  • The toolkit enhances the industrial application potential of B. subtilis as a biological chassis.