Time-resolved imaging-based CRISPRi screening

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Abstract

Our ability to connect genotypic variation to biologically important phenotypes has been seriously limited by the gap between live-cell microscopy and library-scale genomic engineering. Here, we show how in situ genotyping of a library of strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference knockdowns impact the coordination of the replication and division cycles of Escherichia coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation to determine which genes are important for cell cycle control. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes and replication initiation volumes. The technology presented in this study enables genome-scale screens of most live-cell microscopy assays.

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Fig. 1: Assay workflow.
Fig. 2: Analysis.
Fig. 3: Phenotypic data averaged for each genotype.
Fig. 4: Fork distribution plots and time-resolved phenotypes.
Fig. 5: Cell-to-cell phenotypic variation for each genotype.

Data availability

The microscopy images and image analysis output associated with the DuMPLING experiments are publicly available at the Image Data Resource (https://idr.openmicroscopy.org/) under the accession number idr0065. Other data from this study are available from the corresponding author upon request.

Code availability

The code used to analyze the DuMPLING microscopy images and generate figures associated with the DuMPLING experiments is provided as Supplementary Software.

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Acknowledgements

This work was supported by the Knut and Alice Wallenberg Foundation (2017.0291 and 2016.0077), the European Research Council (616047) and the Swedish Research Council (VR)(642-2013-7841 and 2016-06213). We are grateful to I. Barkefors for help with the manuscript and figures, to P. Karempudi for making microfluidic molds and to the Dan Andersson lab for kindly sharing E. coli strains.

Author information

J.E. conceived the DuMPLING method and SeqA application. D.C. developed cloning methods, and designed and made the strain library. J.E. and M.J.L. managed the project. D.F. and M.J.L. developed phenotyping methods. J.L. and M.J.L. developed genotyping methods. D.F. built the microscope. J.L. and M.J.L. performed microscopy experiments. S.Z. and D.F. developed the image analysis pipeline. S.Z. and D.F. analyzed the DuMPLING data. D.J. performed repression measurements and NGS growth rate experiments. J.E., M.J.L., D.C. and D.F. wrote the paper with input from all authors.

Correspondence to Michael J. Lawson or Johan Elf.

Ethics declarations

Competing interests

The DuMPLING technology is patented with European patent no. EP3167061 (B1).

Additional information

Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Fig. 1 Layout of the microfluidic chip.

Image adapted from (Baltekin et al. 201713). The holes punched in the PDMS before bonding it to the PDMS are 2.0 (media in, front channel), 7.0 and 8.0 (waste port, front channel), 5.1 and 5.2 (waste port, back channel), 2.1 and 2.2 (cell loading or waste port, front channel).

Supplementary Fig. 2 Test strain CRISPRi growth assay.

Identical line style indicates three replicate cultures from three different colonies of the same plasmid construct in the test strain. No sgRNA: Negative sgRNA control plasmid pGuide-empty. lacY sgRNA: sgRNA against lacY from plasmid pGuide-P1-lacY. Glucose or lactose was used as supplemental carbon source and dCas9 expression/CRISPRi was induced with aTc (+ aTc). This assay was carried out once.

Supplementary Fig. 3 Comparison of relative abundances of genotypes in DuMPLING and NGS experiments.

Horizontal axis: Relative abundance of each strain as measured by next generation sequencing. (Raw sequence counts given in Supplementary Table 2) Vertical Axis: Relative abundance of each strain in the microfluidic chip. (The number of measurements for each genotype is equal to its number of traps as given in Supplementary Table 1.) Colors of dots indicate the growth rate of each genotype as estimated by NGS.

Supplementary Fig. 4 Quantified genotype probe fluorescence.

Histogram of average cell pixel intensity per trap after background reduction in logarithmic scale. The lower intensity fluorescence peaks are fitted to Gaussians (solid red line) and genotypes are determined when the probe fluorescence signal reaches above 7 times the standard deviations (dashed red lines) of the fitted Gaussians. See the Image analysis subsection on Genotyping in the Methods for details of how to determine genotypes.

Supplementary Fig. 5 Estimating initiation volume.

A. (left) Fork distribution plot of ref strain (same data as Fig. 3e). Horizontal is SeqA-YFP cluster location along the long axis of the cell, vertical is cell size, color indicates the probability of finding a SeqA-YFP foci at a given position along the cell long axis for a given cell size. Initiation size (red dashed line) corresponds to the average of individually tracked replication forks. (right) Estimating initiation size (dashed red line) by fitting an error function (solid red line) to bulk data (blue squares) from the regions |x|>0.44 μm in the forkplot. B. (left) Same as Fig. 2c. (right) Estimating initiation size (dashed red line) by fitting a Gaussian (solid red line) to the relative histogram of single cell initiation sizes. C. Same as Fig. 3f in main text.

Supplementary Fig. 6 Fork plot distributions for replica experiments of library subpool 1.

r stands for the run number, n stands for the number of traps detected for each genotype. Dashed and solid lines as defined in Fig. 3e. The range of the horizontal and vertical axes is the same for each fork distribution and the same as in Figs. 3e and 4g.

Supplementary Fig. 7 Fork plot distributions for replica experiments of library subpool 2.

r stands for the run number, n stands for the number of traps detected for each genotype. Dashed and solid lines as defined in Fig. 3e. The range of the horizontal and vertical axes is the same for each fork distribution and the same as in Figs. 3e and 4g.

Supplementary Fig. 8 Fork plot distributions for replica experiments of library subpool 3.

r stands for the run number, n stands for the number of traps detected for each genotype. Dashed and solid lines as defined in Fig. 3e. The range of the horizontal and vertical axes is the same for each fork distribution and the same as in Figs. 3e and 4g.

Supplementary Fig. 9 Fork plot distributions for replica experiments of library subpool 4.

r stands for the run number, n stands for the number of traps detected for each genotype. Dashed and solid lines as defined in Fig. 3e. The range of the horizontal and vertical axes is the same for each fork distribution and the same as in Figs. 3e and 4g.

Supplementary Fig. 10 Fork plot distributions for replica experiments of library subpool 5.

r stands for the run number, n stands for the number of traps detected for each genotype. Dashed and solid lines as defined in Fig. 3e. The range of the horizontal and vertical axes is the same for each fork distribution and the same as in Figs. 3e and 4g.

Supplementary Fig. 11 Fork plot distributions for replica experiments of library subpool 6.

r stands for the run number, n stands for the number of traps detected for each genotype. Dashed and solid lines as defined in Fig. 3e. The range of the horizontal and vertical axes is the same for each fork distribution and the same as in Figs. 3e and 4g.

Supplementary Fig. 12 Correlations between replica DuMPLING experiments.

Correlation plots for normalized average growth rate (Pearson correlation = 0.86, n=215 different strains where growth rates were estimated in both run 1 and run 2), normalized average birth size (Pearson correlation = 0.78, n=215 different strains where birth sizes were estimated in both run 1 and run 2) and normalized average initiation size (Pearson correlation = 0.74, n=191 different strains where initiation sizes were estimated in both run 1 and run 2) between duplicate experiments. Deviations by more than 0.1 from the straight line where run 1 = run 2 are indicated by names. The number of data points used to estimate each average in the three different panels is given in Supplementary Table 1. Reference control strain (ref) is indicated with red dots.

Supplementary information

Supplementary Information

Supplementary Figs. 1–12 and Notes 1–4.

Reporting Summary

Supplementary Video 1

Example of time-lapse fluorescence imaging; one position with segmented cell outlines (yellow) and detected SeqA-YFP clusters (red). The video is truncated in time compared with the complete experiment.

Supplementary Video 2

Collage of time-lapse phase contrast imaging; from 1/3 of the positions in one experiment. The video is truncated in time compared with the complete experiment.

Supplementary Video 3

Collage of time-lapse fluorescence imaging; from 1/3 of the positions in one experiment. The video is truncated in time compared with the complete experiment.

Supplementary Video 4

Fluorescence time-lapse imaging of three cell traps genotyped as ref traps; images are overlaid with segmented cell outlines (yellow) and detected SeqA-YFP clusters (red). There is 2 min between fluorescence images in the experiment. The video playback rate is 7 fps.

Supplementary Video 5

Fluorescence time-lapse imaging of three cell traps genotyped as dedD traps; images are overlaid with segmented cell outlines (yellow) and detected SeqA-YFP clusters (red). There is 2 min between fluorescence images in the experiment. The video playback rate is 7 fps.

Supplementary Video 6

Fluorescence time-lapse imaging of three cell traps genotyped as hda traps; images are overlaid with segmented cell outlines (yellow) and detected SeqA-YFP clusters (red). There is 2 min between fluorescence images in the experiment. The video playback rate is 7 fps.

Supplementary Video 7

Fluorescence time-lapse imaging of three cell traps genotyped as clpP traps; images are overlaid with segmented cell outlines (yellow) and detected SeqA-YFP clusters (red). There is 2 min between fluorescence images in the experiment. The video playback rate is 7 fps.

Supplementary Video 8

Time-lapse phase contrast imaging of three traps with single-gene clpP knockdown cells; there is 5 min between phase contrast images in the experiment. The video playback is at 7 fps.

Supplementary Video 9

Time-lapse phase contrast imaging of three traps with single-gene clpP knockout cells; there is 5 min between phase contrast images in the experiment. The video playback is at 7 fps.

Supplementary Table 1

DuMPLING data and statistics; estimates of mean and CV of growth rate, birth size and initiation size for each genotype in two replicates of the DuMPLING microscopy experiments (shown in two different tabs, ‘run 1’ and ‘run 2’). Each estimate is normalized to the ref strain in each experiment. The table also includes the number of data points used in each estimate (N cells) and the number of traps (N traps) that each detected genotype occupies. For initiation size bulk, the column ‘N cell detections’ implies the total number of detected SeqA-YFP foci used in fitting the initiation size.

Supplementary Table 2

NGS statistics; the number of occurrences of each strain before (t = 0) and after (t = 12 h) pooled competition is indicated, as well as the corresponding fractional abundances calculated from the numbers of occurrences. The resulting changes in relative abundance and relative fitness are also given, calculated as described in Supplementary Note 3.

Supplementary Table 3

Comparison of DuMPLING experiments with single-gene knockdown and knockouts; for nine selected genotypes. Table includes normalized means, normalized CV and the number of data points used to estimate these (N cells).

Supplementary Table 4

List of genes in each of the transcriptional units of the CRISPRi target genes in the DuMPLING library; the table also includes genes in each transcriptional unit that have been reported essential.

Supplementary Table 5

Sequences of CRISPRi spacers, barcodes and oligonucleotides; for each genotype in the DuMPLING library.

Supplementary Table 6

Strains used in this study; includes genotypes and description.

Supplementary Software

Compressed file with all software used to derive and analyze the DuMPLING data.

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Camsund, D., Lawson, M.J., Larsson, J. et al. Time-resolved imaging-based CRISPRi screening. Nat Methods (2019) doi:10.1038/s41592-019-0629-y

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