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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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Genetic Markers Remain Detectable in Genetically Engineered Microbes Biocontained with a CRISPR Kill Switch.

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Genetically engineered microbes with CRISPR-Cas9 kill switches can still release detectable DNA after biocontainment. This intact microbial DNA poses environmental risks and challenges for monitoring genetically engineered microbes (GEMs).

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Area of Science:

  • Environmental microbiology
  • Synthetic biology
  • Molecular biology

Background:

  • Biocontainment strategies are crucial for genetically engineered microbes (GEMs) in environmental applications.
  • CRISPR-Cas9 kill switches aim to prevent unintended proliferation of GEMs.
  • Residual GEM DNA post-biocontainment presents environmental monitoring challenges.

Purpose of the Study:

  • To assess the effectiveness of a CRISPR-Cas9 kill switch in preventing GEM proliferation and DNA release.
  • To investigate the fate of GEM DNA after biocontainment using a CRISPR-Cas9 system.
  • To evaluate the environmental persistence and detectability of GEM DNA.

Main Methods:

  • Utilized a model Escherichia coli GEM with a CRISPR-Cas9 kill switch.
  • Quantified GEM escape rates using colony forming units (cfu) and CRISPR-targeted gene abundances.
  • Assessed DNA integrity and DNase resistance within 1 hour and over multiple days in different environments.

Main Results:

  • CRISPR-Cas9 kill switch significantly reduced viable GEMs (cfu) but showed high target gene abundance, indicating intact DNA.
  • High escape rates (10-1.6 to 10-1.0) were observed via gene abundance, contrasting with low cfu rates (10-6.2).
  • GEM DNA remained largely intact and DNase-resistant within cells for at least 1 hour, degrading over days in river water.

Conclusions:

  • CRISPR-Cas9 kill switches effectively inhibit GEM growth but do not eliminate detectable DNA.
  • Intact GEM DNA persists after biocontainment, posing risks and complicating monitoring.
  • Further research is needed to understand the full impact of biocontainment on GEMs and their DNA to mitigate environmental risks.