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CRISPR/Cas13d-Mediated Microbial RNA Knockdown.

Kun Zhang1,2, Zhihui Zhang1,2, Jianan Kang3

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

Frontiers in Bioengineering and Biotechnology
|August 28, 2020
PubMed
Summary
This summary is machine-generated.

Researchers developed a CRISPR/Cas13d RNA knockdown platform for bacteria. While inducible expression of CasRx in E. coli showed moderate success, further optimization is needed for practical microbial applications.

Keywords:
CRISPRCas13dCasRxRNA knockdowntype IV-D CRISPR effector

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Area of Science:

  • Molecular Biology
  • Microbial Genetics
  • CRISPR Technology

Background:

  • Type IV-D CRISPR/Cas systems, specifically CRISPR/Cas13d, offer RNA-targeting capabilities for gene knockdown in various cell types.
  • Cas13d's unique RNase activities and protospacer flanking sequence-independent cleavage make it a potential tool for microbial gene regulation.
  • Previous applications of CRISPR/Cas13d have focused on mammalian and plant cells, with limited exploration in bacterial systems.

Purpose of the Study:

  • To establish a functional CRISPR/Cas13d-mediated RNA knockdown platform in bacterial hosts.
  • To investigate the feasibility and efficiency of using the CasRx enzyme for gene repression in bacteria.
  • To overcome challenges associated with CasRx toxicity and optimize its inducible expression in bacterial chassis.

Main Methods:

  • Selection of CasRx from *Ruminococcus flavefaciens* XPD3002 for its high activity.
  • Development of an inducible expression system using a low copy number vector, tightly controlled promoter, and weakened ribosome binding site to mitigate CasRx toxicity.
  • Optimization of inducer concentration, guide RNA (gRNA) design, and target gene expression levels for RNA knockdown.
  • Application of the system to repress green fluorescent protein (GFP) expression in *Escherichia coli*.

Main Results:

  • Successful construction of an inducible CRISPR/Cas13d expression system for CasRx in *E. coli* and *Corynebacterium glutamicum*.
  • Achieved moderate gene repression efficiencies: 30-50% at the protein level and approximately 70% at the mRNA level for GFP.
  • Observed potential interference from Cas13d's collateral cleavage activity on bystander RNAs, impacting overall knockdown efficiency and microbial metabolism.

Conclusions:

  • The developed inducible CRISPR/Cas13d system shows promise for RNA knockdown in bacteria, but efficiency requires improvement.
  • CasRx toxicity and collateral cleavage activity are significant factors affecting its application in microbial systems.
  • Further research is necessary to understand cellular responses to CRISPR/Cas13d and enhance its RNA knockdown capabilities for practical microbial applications.