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Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
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An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus.

Drew M DeLorenzo1, Jinjin Diao1, Rhiannon Carr1

  • 1Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, United States.

ACS Synthetic Biology
|March 31, 2021
PubMed
Summary
This summary is machine-generated.

Researchers developed an improved CRISPR interference (CRISPRi) system for Rhodococcus opacus, enhancing gene repression efficiency to 85% for lignin valorization studies.

Keywords:
CRISPR interferencemetabolic engineeringmetabolic pathway elucidationmuconatenonmodel organism

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Area of Science:

  • Microbiology
  • Synthetic Biology
  • Biotechnology

Background:

  • Rhodococcus opacus is a promising bacterium for lignin valorization.
  • Functional gene studies in R. opacus are hindered by limited genetic tools.
  • Previous CRISPR interference (CRISPRi) systems showed limited repression efficiency.

Purpose of the Study:

  • To develop a more efficient and user-friendly CRISPRi system for Rhodococcus opacus.
  • To facilitate single-gene function studies in this nonmodel organism.
  • To engineer R. opacus for enhanced lignin valorization.

Main Methods:

  • Developed a CRISPRi system utilizing T7 RNA polymerase for small guide RNA expression.
  • Implemented a PCR-free cloning strategy for parallel plasmid construction.
  • Applied the system to confirm roles of four metabolic pathway genes and induce muconate accumulation.

Main Results:

  • Achieved a maximum gene repression efficiency of 85%, a significant improvement over previous systems.
  • Successfully validated the functions of four key metabolic genes.
  • Demonstrated inducible accumulation of muconate, a precursor for nylon production.

Conclusions:

  • The enhanced CRISPRi system provides a powerful tool for functional genomics in R. opacus.
  • This tool facilitates the engineering of R. opacus as a microbial chassis for sustainable chemical production from lignin.
  • The system enables efficient genetic manipulation in GC-rich organisms like R. opacus.