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

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 crucial cellular processes 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 clarify the role of bacterial heterogeneity and population dynamics for gene circuits affecting cell division. Filamentation is shown to strongly increase gene expression variability in the bacteria. Furthermore, we find reversible switching only in a small subpopulation of the bacteria, which takes over the population upon continued cell division. Successful restoration of division occurs faster in the presence of antisense sgRNAs than upon simple termination of CRISPRi induction.