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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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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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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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Genetically stable CRISPR-based kill switches for engineered microbes.

Austin G Rottinghaus1, Aura Ferreiro2,3, Skye R S Fishbein2,4

  • 1Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, USA.

Nature Communications
|February 4, 2022
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Summary
This summary is machine-generated.

We engineered CRISPR-based kill switches in probiotic bacteria for safe living therapeutics. Redundant strategies ensured genetic stability, achieving robust microbial biocontainment in the gut and upon excretion.

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Area of Science:

  • Synthetic Biology
  • Microbial Engineering
  • Genetic Circuits

Background:

  • Living therapeutics require robust microbial biocontainment to ensure safety.
  • Genetic instability of kill switch circuits, particularly under selection pressure, poses a significant challenge for engineered microbes.
  • Evolution of escape mutants is a primary concern for maintaining the efficacy of biocontainment systems.

Purpose of the Study:

  • To engineer and validate CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917.
  • To enhance the genetic stability of these kill switches using multiple parallel strategies.
  • To demonstrate effective microbial biocontainment within a host environment and upon excretion.

Main Methods:

  • Development of two CRISPR-based kill switches: a single-input (chemical-responsive) and a two-input (chemical- and temperature-responsive).
  • Implementation of stability strategies: functional redundancy, SOS response modulation, antibiotic-independent plasmid maintenance, and intra-niche competition.
  • In vivo testing within the murine gut to assess selective killing and biocontainment efficacy.
  • Ex vivo assessment of the two-input switch's efficacy upon microbial excretion.

Main Results:

  • Both engineered kill switches demonstrated selective and efficient killing of engineered Escherichia coli Nissle 1917 within the murine gut.
  • The two-input kill switch additionally provided biocontainment upon excretion from the host.
  • The combination of redundant strategies resulted in robust genetic stability and effective biocontainment of the engineered strains.

Conclusions:

  • CRISPR-based kill switches can be engineered for effective microbial biocontainment in living therapeutics.
  • Parallel strategies significantly enhance the genetic stability of kill switches, mitigating the evolution of escape mutants.
  • This work provides a robust framework for developing stable biocontainment systems for future microbial engineering applications.