Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism

Vink, Jochem N.A.; Martens, Koen J.A.; Vlot, Marnix; McKenzie, Rebecca E.; Almendros, Cristóbal; Bonilla, Estrada; Brocken, Daan J.W.; Hohlbein, Johannes; Brouns, Stan J. J.

doi:10.1016/j.molcel.2019.10.021

Cited by 45 publications

(39 citation statements)

References 86 publications

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“…Furthermore, an AcrIIA22 mutant that is partially defective for nickase activity in vitro (Figure 5D) is ∼1,000-fold less effective at protecting a plasmid from SpyCas9 in vivo (Figure 4G). This indicates that modest changes in nickase activity can have major consequences for plasmid survival, which is consistent with our kinetic race model (Figure 6B) and previous observations that non-linear equilibrium dynamics determine whether an MGE withstands CRISPR-Cas immunity 29 .…”

Section: Discussionsupporting

confidence: 91%

“…To be effective, a CRISPR-Cas system must eliminate its target at a faster rate than the target can replicate 29 . Our findings raised the possibility that AcrIIA22 modifies a target plasmid into a SpyCas9-resistant conformation to win this ‘kinetic race’ against SpyCas9, potentially shifting the equilibrium to favor plasmid persistence instead of elimination.…”

Section: Resultsmentioning

confidence: 99%

“…For example, slowing down Cas9 cleavage could increase the time and probability for escape mutants to arise ( e.g. Cas9 target-site variants 1 , deletion mutants 34 ), allow for additional Acr expression 38,39 , or permit further genome replication to overwhelm CRISPR-Cas immunity 29 . This phenomenon – weak tolerance giving rise to long-term resistance – is reproducibly observed in cases of strong selective pressure.…”

Section: Discussionmentioning

confidence: 99%

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The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

Forsberg

Schmidtke

Werther

et al. 2020

Preprint

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To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all mechanistically characterized Acrs directly bind their cognate Cas protein to inactivate CRISPR immunity. Here, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of Clostridial bacteria and their prophages from the human gut microbiome. Uncovered in a functional metagenomic selection, AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic-acid binding proteins. This homology helped us deduce that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids, rendering them less susceptible to SpyCas9, which is highly dependent on negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and mobile genetic elements.

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Section: Discussionsupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

Forsberg

Schmidtke

Werther

et al. 2020

Preprint

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“…An example of such a search comes from the CRISPR system that relies on normal diffusion in 3D to locate a short sequence within a double stranded chromosome based on homology to the guide RNA. There, a single CRISPR-Cas complex spends hours searching for the correct site (Jones et al 2017;Vink et al 2019). A similar mechanism for HR would be too slow, since a further complication in HR is that the diffusivity of the RecA-ssDNA filament is limited due to its large molecular mass and its tethering to the rest of the chromosome.…”

Section: Introductionmentioning

confidence: 99%

Live cell imaging reveals that RecA finds homologous DNA by reduced dimensionality search

Wiktor

Gynnå

Leroy

et al. 2020

Preprint

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The search for a homologous template is a central and critical step in the repair of DNA breaks by homologous recombination. However, it is still unclear how the search process is carried out within a cell. Here, we image double-stranded break (DSB) repair in living E. coli growing in a microfluidic device and show that two segregated homologous sequences find each other less than 9 min after an induced DSB. To characterize the mechanisms of the search process, we use a new RecA fluorescent fusion that rapidly forms structures after DSBs. Initially, RecA forms a bright cluster at the site of the DNA damage and then extends to form a thin, flexible, and dynamic filament. Based on our observations, we propose a model where the 1D ssDNA-RecA filament stretches along the cell, and the homology search is mediated by diffusion of the repair template that can interact with any segment of the filament. This model reduces the complexity of the search from 3D to 2D and quantitatively predicts that genome-wide search for homology can be finished in minutes, in agreement with our measurements.

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“…Single-molecule localization microscopy (SMLM) is the subcollection of super-resolution techniques in which the fluorescent emission profile, ordinarily referred to as a point spread function (PSF), of a single fluorophore is localized with a precision (~ 5 -40 nm) that can exceed the classical resolution limit by more than one order of magnitude [13][14][15][16]. SMLM is therefore an integral part of STORM and PALM, and has been extensively used in biological research [17][18][19], for example to study DNA transcription [20,21], CRISPR-Cas DNA screening [22][23][24], nuclear pore complexes [25,26], and microtubules [27]. In a conventional fluorescence microscope, a PSF from a single emitter in focus resembles an Airy pattern, which can be approximated by a 2-dimensional Gaussian function.…”

Section: Introductionmentioning

confidence: 99%

Analyzing engineered point spread functions using phasor-based single-molecule localization microscopy

Martens

Jabermoradi

Yang

et al. 2020

Preprint

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The point spread function (PSF) of single molecule emitters can be engineered in the Fourier plane to encode threedimensional localization information, creating double-helix, saddle-point or tetra-pod PSFs. Here, we describe and assess adaptations of the phasor-based single-molecule localization microscopy (pSMLM) algorithm to localize single molecules using these PSFs with sub-pixel accuracy. For double-helix, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations, while for saddle-point or tetra-pod, a novel phasor-based deconvolution approach is used. The pSMLM software package delivers similar precision and recall rates to the bestin-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores. pSMLM substantially improves the localization rate by a factor of 2 -4x on a standard CPU, with 1-1.5·10 4 (double-helix) or 2.5·10 5 (saddlepoint/tetra-pod) localizations/second.

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Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism

Abstract: Highlights d 20 Cascade complexes are required to provide 50% protection d Cascade spends equal time probing DNA (30 ms) and diffusing to a next site d Cas8e dynamically associates with Cascade in cells d CRISPR target search and invader replication compete in a kinetic ''arms race''

Cited by 45 publications

References 86 publications

The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

Live cell imaging reveals that RecA finds homologous DNA by reduced dimensionality search

Analyzing engineered point spread functions using phasor-based single-molecule localization microscopy

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