Materials and Methods

CRISPR-Csx28 forms a Cas13b-activated membrane pore required for robust CRISPR-Cas adaptive immunity

Bacterial Strains and Phages

E.coli C3000 (ATCC 15597) were grown in LB or 1.5% LB agar at 37 °C shaking at 200 rpm. Whenever applicable, media were supplemented with ampicillin (50 μg ml−1) and/or chloramphenicol (15 μg ml−1) to ensure the maintenance of plasmids. C3000 cells were used to propagate MS2 phage (ATCC 15597-B1) according to ATCC recommended handling procedure.

E.coli MG1655 harboring phage λ cI857 bor::kanR (a heat inducible mutant prophage; 48) were grown in LB supplemented with 10 mM MgCl2 and kanamycin (50 µg ml-1) at 30 °C shaking at 200rpm until an OD of 0.4. The temperature was increased to 42 °C to elicit the lytic response of the phage which required ∼2 hours for complete clearance of the medium. λ-phage was then purified using a previously published protocol 49 and stored in SM buffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 8 mM MgSO4) at 4 °C. E.coli Rosetta2 (DE3) pLysS (Novagen) transformants were grown in terrific broth (TB) at 37 °C shaking at 180rpm with ampicillin (50 μg ml−1) and chloramphenicol (15 μg ml−1) to ensure the maintenance of plasmids.

Plasmid Construction

Csx28 and Cas13b plasmids in this study are modified versions of the following Addgene plasmids #89909 and #89906, respectively. These plasmids were modified to include a lac operator sequence which is necessary for functional lac operon induction in the presence of IPTG and suppression in the presence of glucose. Additionally, crRNA spacer sequences were inserted between direct repeats at the BsaI restriction site of Cas13b plasmids. Mutants of Csx28 and Cas13b were generated via site- directed mutagenesis.

Efficiency of Plating (EOP)

λ phage was titrated by ten-fold serial dilutions in phage SM buffer. 2 µl of each serial dilution were spotted onto solidified 0.75% top LBA containing 200 µl E. coli culture (pre-grown in 5 ml LB overnight) in 3ml top LBA. Plates were incubated at 37 °C for 16 hours. Plaques were counted and titer was determined as plaque forming units (pfu) ml−1. EOP was calculated as (pfu ml−1 (test strain)/pfu ml−1 (control strain, E. coli C3000)). All experiments include three individual biological repeats with each including technical replicates in triplicate. Csx28 and Cas13b and plasmid mutants were transformed into E. coli C3000 prior to plating. Data was plotted using GraphPad Prism 9.

One-step phage growth curves

Overnight solutions of E. coli were used to inoculate 20 ml cultures of LB+10mM MgCl2 at a starting OD600 of 0.1 and were grown to OD600 of 0.4 when they were infected with λ phage at an MOI of 0.1. Adsorption was carried out for 5 min, followed by three 20ml washes with LB+10mM MgCl2 to remove “free” unabsorbed phage. Pellets were resuspended in 20 ml LB+10mM MgCl2. For the 0min time point (Efficiency of Center of Infection, ECOI), two 100µl aliquots were taken immediately: one serially diluted in LB+10 mM MgCl2 (untreated sample) and plated directly on top LBA with susceptible host E.coli, and the other 100 µl aliquot was treated with a 2% chloroform LB+10mM MgCl2 solution (treated sample; chloroform lyses the cells and results in only the measurement of mature phage) for 2-3 mins and then serially diluted in LB+10 mM MgCl2 and plated on top LBA with E.coli. Subsequent time points were subjected to only chloroform treatment and titrated in the same manner. At each time point, the pfu ml−1 was determined for each strain with the titer representing the number of infectious centers formed. The ECOI was calculated from the untreated sample at 0 min as (pfu ml−1 (test strain)/pfu ml−1 (control strain, E. coli)). The average first burst size was calculated as (pfu ml-1 (80min treated)/ECOI). All experiments include three individual biological repeats with each including technical replicates in triplicate. Data was plotted using GraphPad Prism 9.

Bacterial Growth Curves

Overnight cultures of bacteria (E. coli with plasmids pEmpty, Cas13b, and/or Csx28) or negative control (E. coli with both empty plasmids) were diluted to a final OD600 0.1 in LB+10 mM MgCl2 supplemented with ampicillin and chloramphenicol in presence of λ phage for a final MOI of 0, 0.2 and 2. 200 μl of the mixed samples were transferred into wells of a 96-well plate in triplicate. Plates were incubated at 37 °C with shaking in a Tecan Spark plate reader with A600 measurements taken every 10 minutes with the first time point at 0 min. Data was plotted using GraphPad Prism 9.

Csx28 protein expression and purification

The Csx28 expression vector was assembled by using a PCR fragment of the PbuCsx28 ORF from the PbuCsx28 Addgene plasmid (#89909; Table S2). Csx28 was C-terminally tagged with a TEV-MBP-His6 cleavage site sequence with expression driven by a T7 promoter. Expression vectors were transformed into Rosetta2 (DE3) pLysS E. coli cells and grown in TB broth at 37 °C, induced at mid-log phase with 0.5 mM IPTG, and grown for an additional three hours until harvested. Cell pellets were resuspended in lysis buffer (50 mM HEPES pH 7.5, 1 M NaCl, 5% glycerol, 1 mM TCEP, 1% DDM, 20mM imidazole, and EDTA-free protease inhibitor (Roche)), lysed by sonication, and clarified by centrifugation at 16,000g. Soluble Csx28-TEV-MBP-His6 was isolated over metal ion affinity chromatography and rinsed with wash buffer (50mM HEPES pH 7.5, 1 M NaCl, 5% glycerol, 1 mM TCEP, 0.1% DDM, and 40mM imidazole) and eluted in elution buffer (50mM HEPES pH 7.5, 1 M NaCl, 5% glycerol, 1 mM TCEP, 0.1% DDM, and 300mM imidazole). Eluate was treated with TEV protease at 4 °C during overnight dialysis in dialysis buffer (50mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, and 1 mM TCEP) to remove the MBP-His6 tag. Cleaved protein was loaded onto a second metal ion affinity column to clear the MBP-His6 tag. Csx28 eluate was concentrated and further purified via size-exclusion chromatography on a GE S200 column in gel filtration buffer (20mM HEPES pH 7.5, 200 mM KCl, 1 mM TCEP, 5% glycerol, and 0.1% DDM) and stored at −80 °C. Size exclusion chromatography traces were plotted using GraphPad Prism 9.

Static light scattering size exclusion chromatography (SEC-SLS)

Purified Csx28 was loaded onto a Superdex 200 increase column (GE Healthcare) equipped with in-line UV (Postnova Analytics), static light scattering and refractive index detectors (Precision Detectors), and equilibrated with a buffer containing 20mM HEPES pH 7.5, 200 mM KCl, 1 mM TCEP, 5% glycerol, and 0.1% DDM. Data were collected using (PrecisionAcquire32 and PrecisionDiscovery32, Precision Detectors), and molecular masses were calculated using manually normalized values and the three detector method 50 using BSA as a known molecular mass control. Data was plotted using GraphPad Prism 9.

Cryo-EM sample preparation

Graphene-oxide (GO) covered grids were prepared following previously published protocols 51, using C-Flat 1.2/1.3 grids (Protochip), and vitrified using a Mark IV Vitrobot (ThermoFisher) at 4°C and 100% humidity. 4 mL of 0.35 mg/mL Csx28 was applied to a freshly prepared GO grid, blotted for 6 seconds and blot force 6, and subsequently plunged into a slurry of liquid ethane cooled using liquid nitrogen.

Cryo-EM image processing and Model building

Prepared grids were imaged at 63,000X nominal magnification (1.33Å/pixel) using Talos Arctica (ThermoFisher) equipped with a Gatan K3 direct electron detector (Gatan) and Gatan bioquantum energy filter (GIF, Gatan). Prior to image acquisition, the microscope was carefully aligned according to recommended procedures for coma-free and parallel illumination settings 52. A total of 904 micrographs were collected using SerialEM 53, 2 by 2 image shift, and nominal defocus range of -1 mm to -2.5 mm. The total dose was 47 electrons/Å2 fractionated into 44 frames and a total of 2.96 seconds exposure (fig. S6A, Table S1). The following pre-processing and micrograph selection steps were carried out using Warp 54: beam-induced motion correction, CTF estimation, and particle picking. The final set of 314 micrographs with an estimated resolution of 5 Å or better were imported to cryoSPARC 55 for subsequent processing. Template-based particle picking in cryoSPARC resulted in 247,026 particles. 2D classification of this particle set resulted in multiple 2D classes of side-like views and one top-view with 8- fold symmetry, resulting in 118,793 selected particles. Ab initio model generation (C1 symmetry) resulted in a reconstruction with clear 8-fold symmetry. Subsequent downstream processing steps were performed while imposing C8 symmetry (fig. S6B- C).

After non-uniform refinement in cryoSPARC, the particle stack was imported to RELION 56 for subsequent refinement. Further rounds of 3D classification resulted in two major classes of Csx28 octamer, and one minor class consisting of two stacked Csx28 octamers (i.e. a dimer of octamers). As two octamer classes didn’t show distinct conformational differences, two classes of 111,831 particles were combined for further refinement. Extensive CTF refinement and Bayesian polishing with RELION 56, 57 was followed. Subsequent rounds of 3D classification to identify classes with qualitatively high-resolution features improved the final resolution from 4.3 Å to 3.65 Å and 58,694 final particles. Local resolution of the final map was estimated using cryoSPARC (fig. S6D). Although this final map showed significantly improved overall resolution, N-terminal trans-membrane helixes were better resolved in the initial cryoSPARC reconstruction (from 118,793 particles before rounds of classification in RELION, fig. S6C). The appearance of the N-terminal helices (7 Å+ resolution) is likely due to the liberal masking imposed by the initial cryoSPARC reconstruction (which also includes micelle density, 10 Å) and the comparatively worse resolution of the N-terminal helices (∼7 Å) compared to that of the soluble portion (4 – 5 Å) of Csx28 (which was subjected to high-resolution refinement).

The final map was sharpened using RELION postprocess function and automatically estimated B-factor (−118 Å2). The sharpened map was of sufficient resolution (3.6 Å) for de novo model building (as homologous structures were not available). The initial atomic model was manually built using coot 58, focusing on a single asymmetric unit. Most of the model (from residue 32 to 171) was built using the final map from RELION, and N-terminal trans-membrane helix (from residue 19 to 31) was built using the initial cryoSPARC reconstruction. The manually built subunit was then manually docked into the density using UCSF Chimera 59 in order to create the octameric assembly. This model was subjected to iterative process of manual inspection, followed by manual adjustments using coot 58, followed by symmetric refinement using RosettaES 60. Model-map FSC (fig. S6E), EMRinger score 61, and MolProbity 62 (Table S1) were calculated using Phenix 63, 64 model validation tools.

Membrane Fractionation and Western blotting

For western blot detection, Csx28 and Cas13b plasmids were modified to contain a C- terminal V5 and N-terminal 3xHA epitope tag, respectively. Fractionation was performed as previously described 65. Briefly, 300ml cultures of pEmpty + pACYC184 (ϕλCas13 empty control), HA-Cas13b + pEmpty, pACYC184 + Csx28-V5, and HA-Cas13b + Csx28-V5 were harvested at OD 0.6, resuspended in 15 ml cell resuspension buffer (50 mM sodium phosphate buffer, 300 mM NaCl, 2 mM MgCl2, 0.2 mg/ml DNaseI, EDTA- free protease inhibitor (Roche) and 0.1 mg/ml lysozyme) and lysed using a French press at 12,000 psi. Lysate was cleared of unbroken cells by centrifugation (12,000 x g) and samples of the supernatant (whole cell lysate) were collected for Western analysis. The lysate was further purified by ultracentrifugation (180,000 x g) which resulted in cytosol (supernate) and membrane (pellet) fractions. The membrane fraction was resuspended in membrane resuspension buffer (50 mM sodium phosphate buffer, 300 mM NaCl, 5% glycerol, EDTA-free protease inhibitor (Roche), 2% DDM) and was further separated into soluble membrane fraction and insoluble membrane fraction via ultracentrifugation (180,000 x g). Samples from the cytosol, soluble membrane, and insoluble membrane fractions were collected for western analysis. Sample loading was normalized by running 0.1% of each total fraction volume. Two gels with identical sample loading order and amount were probed and stripped for subsequent probing. HA-Cas13b was detected using anti-HA antibody (1:1000 in PBST, Roche) and Csx28- V5 was detected by anti-V5 antibody (1:5000 in PBST, Invitrogen). Cytoplasmic control, DnaK, was detected by rabbit anti-DnaK antibody (1:1000 in PBST, Biorybt) and membrane control, OmpC, was detected by anti-OmpC antibody (1:1000 in PBST, Biorybt).

In vivo Protein crosslinking

Protein in vivo crosslinking was carried out with NHS ester, DSS (disuccinimidyl suberate, spacer arm 11.4 Å) (Thermo Scientific) also as suggested by the manufacturer. Briefly, 2×1010 non-infected or λ phage 1 hr-post infected bacterial cells expressing V5-tagged Csx28, HA-tagged Cas13 or combination of both proteins together with C3000 host strain as negative control were washed three times in ice-cold 1×PBS (to remove any amine-containing compounds) and treated with 2 mM DSS for 30 min at room temperature. Then reactions were quenched by addition of 1 M Tris pH 7.5 to a final concentration 20 mM for 15 min at room temperature and cells collected by centrifugation. The status of Csx28 protein in crosslinked cells was determined by making the total cell extracts and carrying out Western blot analysis as described above.

Flow Cytometry

Overnight cultures of E. coli C3000 in LB were diluted to OD 0.1 and grown until OD 0.4, at this time cells were infected with λ- phage at an MOI of 1 and 10 mM MgCl2 was added to the media. 200 µl samples were collected at time 0 min, 30 min, and 90 min post infection. Samples were treated with the fluorescent voltage sensitive dye DiBAC4(3) (5 µM) for 10 minutes at 37 °C. Treated cells were then spun down and washed with fresh LB+10mM MgCl2 twice and finally diluted 1:10 in fresh LB+10mM MgCl2. Samples were measured using a Cytek Aurora Full Spectrum Flow Cytometer measuring 100,000 events (cells) per sample and data were analyzed using FCS Express7 Research (De Novo Software) and data was plotted using GraphPad Prism 9.

Resazurin assays for cellular viability and metabolic activity

Cells were prepared as previously described as above for bacterial growth curves with the exception that assay was paused after 1 hour post lambda infection to allow for resazurin addition. Resazurin (final concentration 3 µg/ml; stock dissolved in PBS, pH7.4 and then 0.2 µm filtered sterilized) was added to each 200 µl sample-containing well in addition to three wells of LB media control, the assay was resumed with fluorescent measurements taken every 10 minutes with 560 nm excitation / 590 nm emission wavelengths using a Tecan Spark plater reader.

Article TitleCRISPR-Csx28 forms a Cas13b-activated membrane pore required for robust CRISPR-Cas adaptive immunity

Abstract

Type VI CRISPR-Cas systems use the RNA-guided RNase Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. Here we show that Csx28, of Type VI-B2 systems, forms membrane pore structures to slow cellular metabolism upon viral infection, and this activity drastically increases anti-viral defense. High- resolution cryo-EM reveals that Csx28 exists unexpectedly as a detergent-encapsulated octameric pore, and we then show these Csx28 pores are membrane localized in vivo. Activation of Csx28 in vivo strictly requires sequence-specific recognition of viral mRNAs by Cas13b, and this activation results in Csx28-mediated membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Together, our work reveals an unprecedented mechanism by which Csx28 acts as a downstream, Cas13b-activated, effector protein that uses membrane perturbation as an anti-viral defense strategy.


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