Conformational Control of Cascade Interference and Priming Activities in CRISPR Immunity  Chaoyou Xue, Natalie R. Whitis, Dipali G. Sashital  Molecular Cell  Volume 64, Issue 4, Pages 826-834 (November 2016) DOI: 10.1016/j.molcel.2016.09.033 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 64, 826-834DOI: (10.1016/j.molcel.2016.09.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Direct Detection of Cse1 Conformational Changes by FRET (A) Full view of apo Cascade structure (PDB: 4TVX). Cse1 NTD, light green; Cse1 CTD, dark green; Cas5, purple; Cse2 subunits, light orange; Cas7 subunits, light blue; Cas6, light red. (B–D) Close-up of Cse1 DNA-binding face (top) and solvent-exposed face (bottom) in apo (B), ssDNA-bound (C), and dsDNA-bound Cascade (D). PDB: 4TVX (B); PDB: 4QYZ (C) with modeled chains from PDB: 4TVX (see Supplemental Experimental Procedures); and PDB: 5H9F (D). The L1 motif is shown in red, and the PAM is shown in yellow in the dsDNA bound structure. Cy3 (green stars) and Cy5 (red stars) labeling sites are indicated, with corresponding distances between sites. See also Table S1 and Movies S1 and S2. (E–G) SDS-PAGE analysis of Cy3 and Cy5 labeling. The gel was analyzed by Coomassie Blue staining (E), Cy3 scan at 560–580 nm (F), or Cy5 scan at ≥665 nm (G). Gel lanes: (1) wild-type (WT) Cse1 or Cse2-Cas6, containing all native Cys residues; (2) minimal-Cys Cse1 or Cse2-Cas6; (3) minimal-Cys N73C Cse1 or Minimal-Cys Cse2-Cas6 containing K169C Cas5. See also Figure S1. (H) Fluorescence emission spectra for individually labeled Cse1 and Cse2-Cas6 and reconstituted FRET-enabled Cascade. Excitation wavelength, 530 nm. (I and J) Fluorescence emission spectra for FRET-enabled Cascade containing Cse1-NTDCy3 (I) or Cse1-CTDCy3 (J) binding to various concentrations of ssDNA (I) or dsDNA (J). The insets show EFRET at various molar equivalents of the DNA substrates; error bar represents mean (n = 3) ± SD. Dashed lines are spectra for Cy3-labeled Cse1 + DNA in the absence of acceptor; solid lines are spectra for FRET-enabled Cascade + DNA. Molecular Cell 2016 64, 826-834DOI: (10.1016/j.molcel.2016.09.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Target Mutations Inhibit Interference through Alteration of Cse1 Conformation (A) Flow cytometry spectra for E. coli cells harboring empty GFP-reporter plasmid (–), GFP-reporter plasmid with targets containing a canonical PAM and fully matching (FM), or seed mismatch (MM; position 1 rG-dG mismatch) protospacer after one growth cycle (8 hr). (B) Flow cytometry spectra for E. coli cells harboring GFP-reporter plasmids containing fully matching protospacer with canonical PAM (AAG) or mutant PAMs (AAA, AGA) after two growth cycles (12 hr each). For (A) and (B), high GFP fluorescence (green) indicates full retention of plasmid, low GFP fluorescence (blue) indicates partial plasmid loss, and GFP− cells (red) indicate complete plasmid loss. (C and D) Primed spacer acquisition against GFP-reporter targets after one 8 hr (C) or two 12 hr (D) growth cycles. CRISPR arrays from genomic DNA were PCR amplified and visualized by gel electrophoresis to detect the acquisition of new spacers (+1 band) relative to original (O) product. (E and F) Quantified interference and priming efficiencies for specified targets after one 8 hr (E) or two 12 hr (F) growth cycles. Interference efficiencies are the percentage of GFP– cells in flow cytometry experiments. Priming efficiencies are quantified from CRISPR amplicons (see Supplemental Experimental Procedures). Error bar represents mean (n = 3) ± SD. (G and H) Changes in EFRET for Cascade containing Cse1-NTDCy3 (G) or Cse1-CTDCy3 (H) upon binding to DNA targets. For each domain, FRET was measured with Cy3 located at two different positions. For (G) and (H), error bar represents mean (n = 3) ± propagated SD (see Supplemental Experimental Procedures). See also Figures S2A–S2C and S2E–S2N. (I) In vitro Cascade-dependent Cas3 cleavage of pUC19 plasmid DNA containing either an FM or MM protospacer (with AAG PAM) analyzed by gel electrophoresis. Cascade was bound to plasmid prior to initiation of Cas3 cleavage, and aliquots were quenched at indicated time points. See also Figure S2D. DNA is labeled as follows: OC, open circle; L, linear; nSC, negatively supercoiled; D, degraded. Molecular Cell 2016 64, 826-834DOI: (10.1016/j.molcel.2016.09.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Cse1 L1 Mutants Switch Immune Response by Promoting the Open Conformation (A) Plasmid loss and spacer acquisition rates for strains expressing WT, F129A, and N131A Cse1 against a GFP-reporter plasmid containing a bona fide target after 8 hr growth. For comparison, WT activity against an AGA target after 8 hr growth is also plotted. WT plasmid loss against the canonical target are reproduced from Figures 2B and 2C. Error bar represents mean (n = 3) ± SD. See also Figures S4A and S4B. (B) In vitro interference assay against plasmid target. Cascade bearing WT, F129A, or N131A Cse1 were bound to DNA prior to initiation of Cas3 cleavage. See also Figures S4C and S4D. (C) Change in EFRET for Cascade containing N131A Cse1-NTDCy3 or N131A Cse1-CTDCy3 upon binding to a canonical dsDNA target with fully matching protospacer and AAG PAM or ssDNA with fully matching protospacer. Cy3 labeling positions: NTD–N73C; CTD–N376C. See also Figures S4E–S4I. (D) Change in EFRET for Cascade containing WT (minimal-Cys) Cse1-NTDCy3 or Cse1-CTDCy3 upon binding to a canonical dsDNA target with fully matching protospacer and AAA PAM or ssDNA with fully matching protospacer. Values are reproduced from experiments shown in Figures 2G and 2H for comparison with (C). For (C) and (D), error bar represents mean (n = 3) ± SD propagated for subtraction (see Experimental Procedures). Molecular Cell 2016 64, 826-834DOI: (10.1016/j.molcel.2016.09.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Model for Conformational Control of Cascade Function (A) When Cse1 adopts the closed conformation, CTD locking leads to recruitment of the Cas3 endonuclease and interference. (B) Alternatively, Cse1 adopts the open conformation, which may promote priming through Cas1-Cas2-dependent recruitment of Cas3 (Redding et al., 2015). The two conformations in (A) and (B) exist in a dynamic equilibrium. Favorable interactions between the PAM-NTD and L1-crRNA promote the closed and locked conformation, while disruption of these interactions through mutation of the PAM, seed, or L1 motif promote the open conformation. Molecular Cell 2016 64, 826-834DOI: (10.1016/j.molcel.2016.09.033) Copyright © 2016 Elsevier Inc. Terms and Conditions