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.
Fig_2b_c_cell-free
This file contains the data from Fig 2. mVenus fluorescence intensities were measured with a plate reader. mVenus was expressed from a purified plasmid in E. coli based cell extract and purified sgRNA, asgRNA and/or dCas9 were supplemented.
Fig_2d_RT-qPCR
This zip-file contains the following files:
"ftsZ -dRFU.xls" - contains the data for the melt curve in Figure S3 (inset).
"Cq ftsZ.xls" - contains the data shown in Figure S3A.
"Cq ref genes.xls" - contains the data used to normalize the data shown in Figure 2D.
"ftsZgenePos.xls" - this contains the position list and sample names for the data in "cq ftsZ" and "ftsZ -dRFU".
"RefgenesPos.xls" - this contain the position list and sample names for the data in "cq ref genes".
Fig_3a_Images
This file contains the phase-contrast microscopy images of the induced cells at different time points used for the histogram of the cell lengths shown in Fig. 3a. The raw data for Fig. 3a are in the excel file “Fig_3a_CellLength.xlsx”.
Fig_3b_Time-series
The phase-contrast bright field images of the data in Fig. 3b. There is a separate file each of the 8 different microscopy stage position. The bacteria in the microfluidic traps at the different microscopy stage positions are exposed to different IPTG/aTc concentrations. An image is taken every 10 minutes from the time point of CRISPRi induction for a total of 100 minutes. The analyzed data is listed in the file "Fig_3_Microfluidics_Cell_length.xlsx".
Fig_4b_Images
Data from Fig 4b. Cell length were measured with the help of NIS-Elements from Nikon from microscopy images. The cells were pipetted from a liquid culture on to a glass slide under the microscope at different time points. Analyzed cell length are listed in file "Fig4_SwitchBach_Boxplot.xlsx".
Fig_4c_FlowCytoData
Flow cytometry data of bacteria being switched to filamentous growth and back to normal growth. Here forward-scattered light (FSC), side-scattered light (SSC) and FL1 (mVenus fluorescence intensity) were measured.
Fig_5a_SwitchBack_Microfluidics
This file contains the data displayed in Fig. 5A and S8. This includes the number of bacteria in the microfluidic traps and the time taken until the first division event after the removal of the CRISPRi inducers.
Fig_S2_cell-free
This file contains the file "Fig_S2_cell-free" which contains the fluorescence intensity values and mVenus concentration from plate reader experiments using purified DNA, dCas9, sgRNA and anti-sgRNA in the PUREexpress and in the homemade cell-free expression system supplemented with T7 RNAP.
Fig_S6_PlateReader
Data from Fig. S5. The absorbance and fluorescence intensity for bacteria with different inducer concentrations was measured for three technical replicates in a plate reader.
Fig_5a_43Ind_SwitchBack_Strategy_i
The microscopy images of the data in Fig. 5a for the following condition: 2 hours of CRISPRi induction (43% induction level) followed by inducers removal in the new medium. There are 35 microscopy stage positions with three channels each: phase-contrast bright field, mVenus, mRFP. The images were binned (2x2).
Fig_5a_43ind_SwitchBack_Strategy_i.zip
Fig_5a_43Ind_SwitchBack_Strategy_iii
The microscopy images of the data in Fig. 5a for the following condition: 2 hours of CRISPRi induction (43% induction level) followed by inducers removal and the addition of 60 nM of AHL in the new medium. There are two data sets with three channels each: phase-contrast bright field, mVenus, mRFP. Data set 1 has 6 microscopy stage positions and data set 2 has 40 microscopy stage positions. The images were binned (2x2).
Fig_5a_100Ind_SwitchBack_Strategy_iii_1stPhase
The microscopy images of the data in Fig. 5a for the following condition: 2 hours of CRISPRi induction (100% induction level) followed by inducers removal and the addition of 60 nM of AHL in the new medium. There are 61 microscopy stage positions with three channels each: phase-contrast bright field, mVenus, mRFP. The images were binned (2x2).
Fig_5a_100Ind_SwitchBack_Strategy_iii_2ndPhase_1
The microscopy images of the data in Fig. 5a for the following condition: 2 hours of CRISPRi induction (100% induction level) followed by inducers removal and the addition of 60 nM of AHL in the new medium. There are 61 microscopy stage positions with three channels each: phase-contrast bright field, mVenus, mRFP. The images were binned (2x2).
Fig_5a_100Ind_SwitchBack_Strategy_iii_2ndPhase_2
The microscopy images of the data in Fig. 5a for the following condition: 2 hours of CRISPRi induction (100% induction level) followed by inducers removal and the addition of 60 nM of AHL in the new medium. There are 61 microscopy stage positions with three channels each: phase-contrast bright field, mVenus, mRFP. The images were binned (2x2).
Fig_S9_S10_SwitchBack_Microfluidics
This file contains the microscopy images of the data in Fig. S9 and S10 for the following condition: 2 hours of CRISPRi induction (43% induction level) followed by inducers removal and the addition of 60 nM of AHL in the new medium. There are 29 microscopy stage positions with three channels each: phase-contrast bright field, mVenus, mRFP. The images were binned (2x2).
readme_Mueckl_et_al
This file includes a list of the data files within this repository and a short description of their content.
