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Quantitative Biology

Quant. Biol.    2017, Vol. 5 Issue (1) : 67-75     DOI: 10.1007/s40484-017-0099-0
Visualization of phage DNA degradation by a type I CRISPR-Cas system at the single-cell level
Jingwen Guan1,2,3,Xu Shi1,2,Roberto Burgos1,Lanying Zeng1,2,3()
1. Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA
2. Center for Phage Technology, Texas A&M University, College Station, TX 77843, USA
3. Molecular and Environmental Plant Sciences, Texas A&M University, College Station, TX 77843, USA
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Background: The CRISPR-Cas system is a widespread prokaryotic defense system which targets and cleaves invasive nucleic acids, such as plasmids or viruses. So far, a great number of studies have focused on the components and mechanisms of this system, however, a direct visualization of CRISPR-Cas degrading invading DNA in real-time has not yet been studied at the single-cell level.

Methods: In this study, we fluorescently label phage lambda DNA in vivo, and track the labeled DNA over time to characterize DNA degradation at the single-cell level.

Results: At the bulk level, the lysogenization frequency of cells harboring CRISPR plasmids decreases significantly compared to cells with a non-CRISPR control. At the single-cell level, host cells with CRISPR activity are unperturbed by phage infection, maintaining normal growth like uninfected cells, where the efficiency of our anti-lambda CRISPR system is around 26%. During the course of time-lapse movies, the average fluorescence of invasive phage DNA in cells with CRISPR activity, decays more rapidly compared to cells without, and phage DNA is fully degraded by around 44 minutes on average. Moreover, the degradation appears to be independent of cell size or the phage DNA ejection site suggesting that Cas proteins are dispersed in sufficient quantities throughout the cell.

Conclusions: With the CRISPR-Cas visualization system we developed, we are able to examine and characterize how a CRISPR system degrades invading phage DNA at the single-cell level. This work provides direct evidence and improves the current understanding on how CRISPR breaks down invading DNA.

Author Summary  The CRISPR-Cas system is a widespread evolutionary adaptation in prokaryotes including archaea and bacteria, defending against invasive nucleic acids, such as plasmids or viruses. We aim to visualize and characterize how a CRISPR system acts within E. coli cells to destroy a phage invader at the single-cell level. By fluorescently labeling and tracking phage lambda DNA after infection using microscopy, we find that CRISPR rapidly degrades phage DNA to allow the cell to live on, and discover some parameters accounting for the cell-to-cell variability of the CRISPR functions, providing insights on how CRISPR systems protect bacteria.
Keywords bacteriophage lambda      CRISPR-Cas      fluorescence microscopy      single-cell analysis      type I CRISPR     
Corresponding Authors: Lanying Zeng   
Issue Date: 22 March 2017
 Cite this article:   
Jingwen Guan,Xu Shi,Roberto Burgos, et al. Visualization of phage DNA degradation by a type I CRISPR-Cas system at the single-cell level[J]. Quant. Biol., 2017, 5(1): 67-75.
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Jingwen Guan
Xu Shi
Roberto Burgos
Lanying Zeng
Fig.1  CRISPR system reduces lysogenization efficiency.

(A) The lysogenization frequencies of CRISPR versus control system for our phage DNA reporter strain at a range of APIs (low API of 1 – 6 and high API of 15 – 65). The control spacer: blue cross marker with blue lines as the mean for low and high APIs; the CRISPR spacer: red circle with red lines as the mean of the low and high APIs. (B) The lysogenization efficiency of the CRISPR system, defined as the ratio of average lysogenization frequency of the CRISPR system relative to that of the control system for low and high APIs. Error bar represents S.D.

Fig.2  CRISPR system apparently degrades labeled phage DNA in single cells.

The DNA of the fluorescently labeled infecting phage (red spot, pointed by purple arrows) is ejected into the host E. coli cell forming a fluorescent spot (green spot, pointed by yellow arrows) at 0 min. The phage DNA (green spot) intensity decreases over time and finally disappears. (A) A lytic cell in a control movie. The green or yellow spot (yellow color is a result of overlay by green and red fluorescence) indicating the phage DNA disappears around 110 min. At 120 min, the cell lyses. (B) A lytic cell in a CRISPR movie. The green or yellow spot indicating the phage DNA disappears around 120 min or persists until cell lysis. At 120 min, the cell lyses. (C) A CRISPR cell (top) and uninfected cell (bottom) in a CRISPR movie. The green spot indicating the phage DNA in the CRISPR cell disappears around 45 min, which is much earlier than that in the lytic cell in (A) and (B). At 70 min, the cell divides, similar to that of the uninfected cell.

Fig.3  Phage DNA intensity decreases faster in CRISPR-active cells.

(A) In the CRISPR movies, the phage DNA intensities of the CRISPR cells (red line, N= 167) decrease much faster than those of the lytic cells (blue line, N= 423) indicating CRISPR is actively functioning to degrade the invading phage DNA. The averages are shown as the thick lines. (B) The histogram of phage DNA spot disappearance time corresponding to the degradation time for CRISPR cells is well fitted to a Gaussian distribution (red line). The time to totally degrade phage DNA is around 43.9±1.5 minutes. (C) The histogram of phage DNA spot disappearance time accounting for the photobleaching and/or phage DNA packaging into the phage head. Around 50% of the lytic cells still have the phage DNA spot at the end of the movies (120 min). (D) The phage DNA spot disappearance time is correlated with cell lysis time with a correlation coefficient of 0.91, p-value of 0.01. Error bar represents S.E.M.

Fig.4  Phage DNA degradation correlates with spot intensity, not with cell size or initial DNA location.

(A) The complete phage DNA degradation or spot disappearance time positively correlates with the maximum intensity of the spot at the beginning of the movie with a correlation coefficient of 0.97, p-value of 0.03. The binned data were obtained with a bin interval of 1.5±105 A.U. (B) The spot disappearance time does not change with the initial cell size with a correlation coefficient of-0.55, p-value of 0.45. The binned data were obtained with a bin interval of 1 mm. (C) The efficiency of CRISPR is very similar for the initial invading phage DNA at polar/mid-cell (0.27±0.04) or non-polar (0.25±0.02) positions with a p-value of 0.02. The diagram of the cell is shown on the top right. (D) The spot disappearance time does not seem to correlate with the initial phage DNA location showing similar disappearance time for polar/mid-cell (42.1±2.4 min) or non-polar (44.7±1.8 min) cell location with a p-value of 0.02. Error bar represents S.E.M.

Bacterial strains, plasmids, and phages
Strain name Relevant genotype Source/Ref.
Bacterial strains
BA16 MG1655, dam-, seqA-yfp, CmR [22]
LZ1436 BA16[pWUR397A, pWUR400, pWUR477], AmpR, StrR, CmR This work
LZ1437 BA16[pWUR397A, pWUR400, pWUR478], AmpR, StrR, CmR This work
Phage strains
lLZ760 Fully methylated, gpD-mosaic, lD-mTurquoise2cI857 bor::KanR [28]
pWUR397A cas3 in pRSF-1b [21]
pWUR400 casA-casB-casC-casD-casE in pCDF-1b [20]
pWUR477 Non-targeting CRISPR/ spacers from E. coli K12 with no homology to phage lambda in pACYCDuet-1 [20]
pWUR478 Template CRISPR/ template strand of lambda genes J, O, R and E in pACYCDuet-1 [20]
Tab.1  The bacteria, plasmids and phages used in this study.
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