Article Figures & Data
Figures
- FIG 1
CRISPRi systems to downregulate gene expression in Caulobacter crescentus. (A) Design of the different constructs used in this study. Each system consists of a nuclease-dead variant of the cas9 gene (dcas9) expressed under the control of a xylose (or vanillate) promoter and the corresponding specific single guide RNA (sgRNA) constitutively expressed (see Materials and Methods). (B) Efficiency of downregulating ctrA expression using the different CRISPRi systems from S. pyogenes, S. thermophilus CRISPR3, and S. pasteurianus. Shown are serial dilutions of each strain on PYE supplemented with 0.2% glucose or 0.3% xylose after 48 h at 30°C. Note that the commonly used S. pyogenes system is not functional in C. crescentus, although it targets the same sequence as the S. pasteurianus system here. Strand and sequence targeting of the different sgRNAs is detailed in Fig. S1A. (C) Strand and sequence targeting of the different sgRNAs for gcrA used in panel D. TSS, transcription start site; T, template; NT, nontemplate. (D) Efficiency of downregulating gcrA expression when targeting the nontemplate strand using dCas9 derived from S. thermophilus CRISPR3 and S. pasteurianus. Shown are serial dilutions of each strain on PYE supplemented with 0.2% glucose or 0.3% xylose after 48 h at 30°C. (E) Efficiency of downregulating ctrA expression with the dCas9 of S. thermophilus CRISPR3 under a vanillate-inducible promoter. Shown are serial dilutions of each strain on PYE or PYE supplemented with 500 μM vanillate after 48 h at 30°C.
- FIG 2
Downregulation of ctrA and gcrA expression in asynchronous populations of cells using the Sth3 CRISPRi system. (A) ctrA (left) and gcrA (right) mRNA levels measured by qRT-PCR and normalized to rpoA levels. (B) CtrA (left) and GcrA (right) protein levels with and without induction of the CRISPRi system. Graphs show quantifications of CtrA or GcrA protein band intensity normalized to RpoA protein band intensity. (C) Flow cytometry profiles showing the DNA content of a mixed population of cells at different time points after induction or not of the ctrA-targeting CRISPRi system. (D) Phase-contrast images of cells 120 (top) or 240 (bottom) min after induction or not of dcas9 alone or in combination with the ctrA- or gcrA-sgRNA, as indicated. Bar, 2 μm.
- FIG 3
Repression of ctrA expression by CRISPRi leads to misregulation of CtrA-regulated genes. Expression changes of CtrA-regulated genes in cells expressing dcas9 and the ctrA-sgRNA (+ xyl) compared to the noninducing condition (+ glu). Column 1, after 2 h by RNA-seq; columns 2 to 4, at t = 0, after 2 h, or after 4 h by microarray. As a control, expression changes of CtrA-regulated genes in the ctrA(V148F)ts strain compared to wild type (wt) are shown in columns 5 to 7 (23, 24).
- FIG 4
Downregulation of ctrA and gcrA expression in synchronized cells. (A) Schematic of CtrA and GcrA abundance during the Caulobacter cell cycle. (B) Normalized ctrA expression in synchronized cells after induction of the CRISPRi system at t = 0 min after synchronization or t = 20 min before synchronization. (C) CtrA protein levels in synchronized cells after induction of CRISPRi system at the same times as indicated for panel B. (D) Flow cytometry profiles after SYTOX staining showing DNA content of synchronized cells when targeting ctrA. (E) Normalized gcrA expression in synchronized cells after induction of the CRISPRi system at t = 20 or 40 min before synchronization. (F) GcrA protein levels in synchronized cells after induction of CRISPRi system at the same times as indicated for panel E. (G) Flow cytometry profiles after SYTOX staining showing DNA content of synchronized cells when targeting gcrA.
- FIG 5
Dual targeting of cpaA and blaA with CRISPRi. (A) Assay to assess sensitivity to phage ΦCbK. Downregulation of the prepilin peptidase gene cpaA confers resistance to the ΦCbK. Phage dilutions were spotted on a lawn of C. crescentus on PYE supplemented with kanamycin and 0.2% glucose or 0.3% xylose. (B) Growth inhibition assay to assess sensitivity to carbenicillin. Downregulation of the β-lactamase gene blaA enhances sensitivity to carbenicillin. Pictures on the left show the zone of growth inhibition (dark zone) surrounding carbenicillin-soaked disks for each indicated strain grown on PYE supplemented with kanamycin and 0.2% glucose or 0.3% xylose. The graph on the right represents the average diameter of inhibition normalized to wild type (WT), measured on n = 6 replicates from n = 2 biological replicates for each strain.
Supplemental Material
FIG S1
Controls for assessing CRISPRi systems to downregulate gene expression in Caulobacter crescentus. (A) Strand and sequence targeting of the different ctrA-sgRNAs used in Fig. 1B. TSS, transcription start site; T, template; NT, nontemplate. (B) Serial dilutions of the ctrA401ts control spotted on PYE plates incubated for 48 hours at 30°C or 37°C where indicated. (C) Expression of the sgRNAs targeting ctrA or gcrA do not affect cell viability in cells lacking the dCas9 protein. Shown are spotting of serial dilutions of each strain on PYE supplemented with 0.2% glucose or 0.3% xylose after 48 hours at 30°C. (D) Serial dilutions of the ΔgcrA Pvan-gcrA strain spotted on PYE plates supplemented with 500 μM vanillate where indicated and incubated for 48 hours at 30°C. Download FIG S1, TIF file, 2.6 MB.
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FIG S2
Loss-of-function phenotypes for cells lacking CtrA or GcrA. (A) Flow cytometry profiles showing the DNA content of a mixed population of ctrA401ts cells at different time points. t = 0 corresponds to when cells were split for growth at 30 and 37°C. (B) Phase-contrast images of cells 120 min after shifting to 37°C for the ctrA401ts strain (top) or after washing away the vanillate for the ΔgcrA Pvan-gcrA strain (bottom), as indicated, leading to cell elongation in both cases. Bar, 2 μm. Download FIG S2, TIF file, 2.3 MB.
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FIG S3
Off-target effects of the Sth3 CRISPRi system. Log2 fold changes in gene expression for the ctrA-sgRNA strain in xylose versus glucose compared to the dcas9-only strain in xylose versus glucose. The effects on ctrA (pink) and the 40 genes that exhibit the largest gene expression changes following induction of ctrA-sgRNA (orange) are shown (Table S1). The dashed line demarcates where gene expression changes are equal between the ctrA-sgRNA and the dcas9-only strain. Genes that are induced or repressed in the ctrA-sgRNA strain but lie along the dashed line are genes whose expression changes are dependent on the shift from glucose to xylose. Download FIG S3, TIF file, 2.7 MB.
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TABLE S1
Top 40 most affected genes following CRISPRi downregulation of ctrA. Download Table S1, XLSX file, 0.02 MB.
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TABLE S2
Analysis of sgRNA target sequences. Download Table S2, DOCX file, 0.02 MB.
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TABLE S3
Strains, plasmids, and primers used. Download Table S3, DOCX file, 0.02 MB.
Copyright © 2020 Guzzo et al.
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TABLE S4
List of all Caulobacter nontemplate-strand sgRNAs. Download Table S4, XLSX file, 0.6 MB.
Copyright © 2020 Guzzo et al.
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