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

Matthaeus Schwarz-Schilling,Andrea Mückl,Friedrich C. Simmel,Katrin Fischer

doi:10.1371/journal.pone.0198058

Matthaeus Schwarz-Schilling, Andrea Mückl + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0198058

Copy DOI

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.

Highlights

The single guide RNA (sgRNA) are induced by IPTG (Isopropyl-β-D-thiogalactopyranoside) via T7 RNA polymerase and were encoded on the ‘CRISPR interference (CRISPRi) plasmid’ together with TetR-controlled dCas9 and mVenus reporter protein
We investigated three switching strategies to revert filamentous cells back to normal growth (Fig 4A): i) passive switching by stopping the production of dCas9-sgRNAs via removal of CRISPRi inducers aTc and IPTG (Fig 4D); ii) by induction of anti-sgRNAs in the presence of CRISPRi inducers, or iii) by removal of CRISPRi inducers and addition of anti-sgRNA inducer
To implement the circuit in viable bacteria, decoy-binding sites for the FtsZ-suppressing dCas9-sgRNA complexes had to be introduced as genetic buffer elements

Summary

Introduction

We use single-cell fluorescence microscopy experiments from which we derive bacterial length distributions [23], growth and division rates, as well as reporter gene expression levels. Transformation of bacteria with the CRISPRi plasmid resulted in filamentous cells even without induction of the expression of dCas9 and sgRNA. After induction of the CRISPRi mechanism with aTc and IPTG, bacterial cells rapidly stopped division and started the expression of mVenus (which was under the control of a pLTetO promoter).

Results

Conclusion