Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.

Experimental Model and Subject Details Microbes

Escherichia coli cells were cultured in LB medium.

Method Details Cloning, expression, and purification The expression and purification of T. fusca Cascade was adapted from a previous protocol ( Huo et al., 2014 ) with some modifications. Specifically, cse1 and the cse2-cas7-cas5e-cas6e gene cassette were cloned into a modified Twin-Strep-SUMO-pET19b vector (Amp R ) and a pCDFDuet-1 vector (Str R ), respectively. A synthetic CRISPR locus containing 4 identical copies of the repeat-spacer sequences was cloned into the pRSFDuet-1 vector (Kan R ). All three plasmids were sequence verified and co-transformed into E. coli BL21 (DE3) star cells. The cell culture was grown in LB medium at 37°C until the optical density at 600 nm reached 0.8. Expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM at 25°C overnight. Cells were harvested by centrifugation and lysed by sonication in buffer A containing 50 mM HEPES pH 7.5, and 500 mM NaCl. The lysate was centrifuged at 15,000 rpm for 60 min at 4°C, and the supernatant was applied onto the pre-equilibrated Strep-Tactin Superflow resin (IBA, Göttingen, Germany). After washing with 100 mL of buffer A. The protein was eluted with buffer B (50 mM HEPES pH 7.5, 500 mM NaCl, and 5 mM desthiobiotin), and the eluted proteins were incubated with SUMO-protease at 4°C overnight. The Tfu Cascade was further purified with size-exclusion chromatography (SEC, HiLoad 16/60 Superdex 200; GE Healthcare) equilibrated with buffer C (10 mM HEPES pH 7.5, 150 mM NaCl, 5 mM DTT), the peak fractions were pooled and snap-frozen in liquid nitrogen for later usage ( Figure S1A ). The assembly of Tfu Cascade/R-loop and Tfu Cascade/seed-bubble is described in detail in the results section. The sequences of the oligos used in preparing DNA substrates for biochemical and structural studies are documented in the Key Resources Table .

Electrophoretic mobility shift assay

The protospacers with an AAG PAM were cloned into the pCDFDuet-1 vector between the BamHI and XhoI sites. The substrates were PCR amplified from the plasmids by using the fluorescent T7 primers and subsequently gel-purified. The dsDNA substrates produced this way are 276-bp in length, with a 5′-6FAM label at the non-target strand. DNA binding was carried out in 20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, and 5% glycerol. 3 nM of dsDNA was incubated with titrations of Tfu Cascade at 60 °C for 30 min. EMSA was performed at 4 °C on 2% agarose gels. Fluorescent signals were recorded using a Typhoon 9200 scanner.

Chemical and enzymatic probing analysis

The protocol for chemical probing with KMnO 4 was modified from the previous publication ( Jore et al., 2011 ). In detail, 3 nM of the 276-bp dsDNA substrates containing a 5’-6FAM label at the non-target strand was incubated with 50 nM Cascade in the 70 µl binding buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, and 5% glycerol. After incubation at the indicated temperatures for 30 min, 20 µl of each reaction was aliquoted for EMSA. The remaining 50 µl was supplemented with 5.5 µl of 16 mM KMnO4, and incubated at 30 °C for 2 min. The permanganate modification was stopped by the addition of 6.5 µl of β-mercaptoethanol and 6.5 µl of 500 mM EDTA. After phenol-chloroform extraction and ethanol precipitation, the nucleic acid pellets were resuspended in 50 µl of 10% piperidine and incubated at 90 °C for 30 min to induce cleavage at the modified residues. For enzymatic probing with P1 nuclease (US Biological) or S1 nuclease (Thermo Scientific), 40 µl of the Cascade–DNA complexes was supplemented with 10 µl reaction buffer (200 mM sodium acetate pH 5.3, 10 mM ZnSO 4 ), and incubated with 1 unit of nuclease at 25 °C for 30 min. The cleavage reactions were stopped by the addition of EDTA to a final concentration of 20 mM. After phenol-chloroform extraction and ethanol precipitation, the pellet was resuspended and separated on a 10% denaturing polyacrylamide gel. The size markers were PCR amplified individually from the same dsDNA substrates using the same 5′-6FAM labeled forward primer and a reverse primer starting from different positions along the substrate. Fluorescent signals were recorded using a Typhoon 9200 scanner. Cascade-mediated Cas3 DNA cleavage assay The Tfu Cas3 protein was expressed and purified following the established protocol ( Huo et al., 2014 ). The dsDNA substrate was PCR generated from 5’-fluorescently labeled primers. The pre-formed Tfu Cascade/R-loop complex was mixed with 100 nM of Tfu Cas3 in a cleavage buffer containing 10 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl 2 and 100 µM CoCl 2 , and the reaction was incubated at 55 °C for 30 min. Afterwards, nucleic acids were phenol-chloroform extracted and separated on a 10% denaturing polyacrylamide gel. Fluorescent signals were recorded on a Typhoon 9200 scanner. EM data acquisition 2.5 µl of ~0.8 mg/ml SEC-purified Cascade complexes were applied to a glow-discharged Quantifoil holey carbon grid (1.2/1.3, 400 mesh). Grids were blotted for 2.5 s at ~85 % humidity and plunge-frozen in liquid ethane using a Cryoplunge 3 System (Gatan). Cryo-EM images were manually collected on a TF30 Polara electron microscope (FEI company) operated at 300 kV and equipped with a K2 Summit direct electron detector (Gatan), as previously described with minor modifications ( Ru et al., 2015 ). The total exposure time of each movie stack was 7.2 s, leading to a total accumulated dose of 49.2 electrons per Å 2 fractionated into 36 frames (200 ms per frame).

Image processing

EM image processing was carried out as previously described with minor modifications ( Ru et al., 2015 ). Dose fractionated super-resolution movie stacks collected from the K2 Summit direct electron detector were binned 2 × 2 to a pixel size of 1.238 Å, and then subjected to motion correction using MotionCor2. A sum of all 36 frames of each movie stack was calculated following a dose-weighting scheme ( Grant and Grigorieff, 2015 ), and used for all following steps of image processing except defocus determination. Defocus values were calculated with the sum of all movie frames without dose weighting, using the program CTFFIND3 ( Mindell and Grigorieff, 2003 ). 2D classification, 3D classification and 3D refinement were carried out using RELION ( Scheres, 2012 ). All refinements followed the gold-standard procedure, in which two half data sets were refined independently. RELION ‘post-processing’ was used to estimate resolution based on the Fourier shell correlation (FSC) = 0.143 criterion after correcting for the effects of a soft shape mask using high-resolution noise substitution ( Chen et al., 2013 ). The overall resolution of the open seed-bubble cryo-EM map (3.8 Å) was estimated based on the FSC = 0.5 criterion instead, to better match the map quality and the validation FSC curves using half data-refined maps and atomic model. Local resolution variations were estimated from two half data maps using ResMap ( Kucukelbir et al., 2014 ). Amplitudes of the final maps were corrected by applying a negative B-factor using RELION ‘post-processing’. The detailed data processing and refinement statistics for the two cryo-EM structures is summarized in Table S1 .

Supplementary Material movie s1 Movie S1, related to Figure 2. Rotating view of the cryo-EM reconstruction of the Tfu Cascade/full R-loop complex Densities were sharpened to review the high-resolution structure features. movie s2 Movie S2, related to Figure 5. Important structure features in the cryo-EM reconstruction of the Tfu Cascade/seed sequence bubble complex The disordered non-target DNA strand and the dsDNA following the seed-bubble were modeled to give perspectives. Note that because the conformational changes in Tfu Cascade have not taken place, the target strand-binding canyon is open and accessible. s1 Supplemental Figure S1, related to Figure 1. Biochemical reconstitution of Type I-E CRISPR interference from T. fusca A, B. Size-exclusion Chromatography and SDS-PAGE profile of purified Tfu Cascade. Red and black traces correspond to 260 and 280 nm UV absorptions, respectively. C. Native EMSA analysis of Tfu Cascade and dsDNA target interaction at 58 °C, with an apparent binding constant of ~ 15 nM. D. PAM preference by Tfu Cascade as revealed by native EMSA. E, F. Cas3-mediated cleavage pattern on Tfu Cascade-bound DNA target, at the non-target (5’-6FAM labeled) and target (5’-Cy5 labeled) DNA strands, respectively. G. Nucleotide-resolution mapping of Cas3 nicking sites on the non-target DNA strand. H, I. Temperature-dependent R-loop formation behavior by Tfu Cascade, as probed by KMnO 4 footprinting and native EMSA, respectively. J. EMSA migration differences likely reflect different extents of DNA bending before and after R-loop formation. s2 Supplemental Figure S2, related to Figure 2. Cryo-EM reconstruction of Tfu Cascade/full R-loop complex A. Representative cryo-EM image and B. 2D averages of Tfu Cascade/full R-loop particles. C. 3D classification and refinement procedures. Three types of 3D reconstructions reached < 4.0 Å resolution. They differ by the presence/absence of PAM-distal dsDNA density, and the density features also differ slightly at the non-target strand gap region (red circles). The final 3D refinement after combining the cryo-EM particles from all good classes generated a density map at 3.3 Å resolution. D. Two different views of the cryo-EM density maps refined using the particles representing the conformations of type 1, 2, and 3. All three maps are low-pass filtered to 6 Å resolution. Major density feature differences are highlighted in the red circles. E. Final 3D reconstruction (left) and its cross-sectional view (right), colored according to local resolution. F. Histogram of voxels with different local resolution. G. Gold-standard FSC curves between two half maps that were calculated from two half data sets (in red), between the summed map and the final atomic model (in blue), between half map 2 and the atomic model refined against half map 1 (in green). H. Euler angle distribution of cryo-EM particles for calculating the final EM map. The height of each bar indicates the number of particles in a particular orientation. s3 Supplemental Figure S3, related to Figure 3, 5. Representative cryo-EM densities in the Tfu Cascade/full R-loop structure A. Contacts for PAM recognition. B. Gln-wedge insertion to unwind DNA from PAM-proximal region. C. Thumb insertion by every Cas7 subunit that disrupts the crRNA-target DNA pairing at every 6 th position. s4 Supplemental Figure S4, related to Figure 3, 8. Comparison between Tfu Cascade/full R-loop and Eco Cascade/R-loop mimic structures A. Overall structure alignment between Tfu Cascade/full R-loop (colored according to Figure 2 ) and Eco Cascade/R-loop mimic (PDB: 5H9E, proteins in blue, nucleic acids in magenta) structures. Major structural differences found in the Tfu Cascade/full R-loop structure are enumerated in text. B. Structure alignment of Cse1 in two structures. Note domain orientation differences as well as different structure features in Cse1_NTD. C. Structure alignment of Cse2. Tfu Cse2 contains a large flexible internal loop and an extension at the C-terminal helix. D. Structure alignment of Cas7. Note the finger domain differences, the K-vise location difference, and the K-Rim location in Tfu Cas7. E. Structure alignment of Cas5e. F. Structure alignment of Cas6e. The two Cas6e structures are superimposable because the Eco Cas6e structure was rigid-body docked into the low-resolution Tfu Cascade/full R-loop EM density without further refinement. Note the differences in the spacer region of the crRNA, due to a slightly extended Cas7 backbone conformation in Tfu Cascade/full R-loop structure. s5 Supplemental Figure S5, related to Figure 5. cryo-EM reconstruction of the Tfu Cascade/seed-bubble complex A. Representative cryo-EM image and B. 2D averages of Tfu Cascade/seed-bubble particles. C. 3D classification and refinement procedures. The reconstructions from the class V (“type 1” conformation) and class VI (“type 2” conformation) in the last round of 3D classification are essentially identical except for the length of the target DNA strand density. The final 3D refinement after combining the cryo-EM particles from both good classes generated a density map at 3.8 Å resolution. D. Two different views of the cryo-EM density maps refined using the particles representing the conformations of type 1, type 2 or both types. All three maps are low-pass filtered to 6 Å resolution. E. Final 3D reconstruction (left) and its cross-sectional view (right), colored according to local resolution. F. Histogram of voxels with different local resolution. G. Gold-standard FSC curves between two half maps that were calculated from two half data sets (in red), between the summed map and the final atomic model (in blue), and between half map 2 and the atomic model refined against half map 1 (in green). H. Euler angle distribution of cryo-EM particles for calculating the final EM map. The height of each bar indicates the number of particles in a particular orientation. table S1 Table S1, related to Methods. Statistical analysis of the cryo-EM structures presented in this study