Sep 12, 2018

Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach

PloS One
Andrea MücklFriedrich C Simmel

Abstract

CRISPR interference (CRISPRi) using dCas9-sgRNA is a powerful tool for the exploration and manipulation of gene functions. Here we quantify the reversible switching of a central process of the bacterial cell cycle by CRISPRi and an antisense RNA mechanism. Reversible induction of filamentous growth in E. coli has been recently demonstrated by controlling the expression levels of the bacterial cell division proteins FtsZ/FtsA via CRISPRi. If FtsZ falls below a critical level, cells cannot divide. However, the cells remain metabolically active and continue with DNA replication. We surmised that this makes them amenable to an inducible antisense RNA strategy to counteract FtsZ inhibition. We show that both static and inducible thresholds can adjust the characteristics of the switching process. Combining bulk data with single cell measurements, we characterize the efficiency of the switching process. Successful restoration of division is found to occur faster in the presence of antisense sgRNAs than upon simple termination of CRISPRi induction.

  • References34
  • Citations2

References

  • References34
  • Citations2

Citations

Mentioned in this Paper

Bacterial Proteins
Alkalescens-Dispar Group
CRISPR-Cas Systems
Filamentous Growth
Gene Editing
Cell Division
Antisense RNA
Cell Cycle
Gene Function
Metabolic Inhibition

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