Publication: Direct visualization of native CRISPR target search in live bacteria reveals Cascade DNA surveillance mechanism

J.N.A. Vink, K.J.A. Martens, M. Vlot, R.E. McKenzie, C. Almendros, B. Estrada Bonilla, D.J.W. Brocken, J. Hohlbein, S.J.J. Brouns, Molecular Cell, 2019, [link]

CRISPR-Cas systems encode RNA-guided surveil-lance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism andkinetics of target search and its impact on CRISPRprotection levels have remained unknown. Here, wevisualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunitCas8e, Cascade spends half its search time rapidly probing DNA (30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and affect CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.

Graphical abstract

News: Welcome to Šarūnė Ivanovaitė and Elmar van der Wijk

Šarūnė recently started her Erasmus+ internship in the group. She will characterise a variety of fluidic devices that we got our hands on including devices for high throughput smFRET screening, trapping of bacteria and establishing DNA curtains. Elmar started his MSc thesis and will utilise new DNA constructs for smFRET based studies of ARF transcription factor binding.

News: Welcome to Abbas Jabermoradi, Ben Tumulero, Vincent Boerkamp, and Sven Koens

Great to see so many people joining the group. Abbas started his PhD thesis on super-resolution based localisation of biomolecules at food-related interfaces. Ben (BSc thesis, BIP) started illuminating bacteria using the PAINT technique. Vincent (MSc thesis, BIP) supports Koen in his quest to monitor particle diffusion in meat replacers. Last but not least, Sven (BSc thesis with BIC & BIP) will study the influence of control elements to the transcription factor binding.

Some PAINT data by Ben

Sample1_celldivision_+BF

News: Welcome to Suyeon Yang and George Vogelaar

Suyeon joined the group in November 2018 for her PhD thesis. She will study lipid oxidation at oil/water interfaces using super-resolution microscopy. George just started his MSc thesis to study diffusion of dCas9 in Lactococcus lactis using single-particle tracking PALM.

News: The miCube moved to Github

Our open (single-molecule) microscopy project, the #miCube, is now on Github [link]!  The page shows a detailed and updated overview of the components, some information on phasor-based SMLM, and many links to similar open hardware projects.

 

News: Welcome to Meike Kronenberg and farewell to Carel Fijen

Meike joined the group for her BSc thesis and will work on DNA polymerase beta. Carel successfully defended his PhD thesis in March and decided to continue working on DNA polymerase beta by joining the Sweasy lab at Yale as a postdoctoral research assistent. All the best and we hope receiving new stocks soon!