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Link to original content: https://pubmed.ncbi.nlm.nih.gov/24505130
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Comparative Study
. 2014 Mar 14;343(6176):1247997.
doi: 10.1126/science.1247997. Epub 2014 Feb 6.

Structures of Cas9 endonucleases reveal RNA-mediated conformational activation

Martin Jinek  1 , Fuguo Jiang, David W Taylor, Samuel H Sternberg, Emine Kaya, Enbo Ma, Carolin Anders, Michael Hauer, Kaihong Zhou, Steven Lin, Matias Kaplan, Anthony T Iavarone, Emmanuelle Charpentier, Eva Nogales, Jennifer A Doudna
Affiliations
Comparative Study

Structures of Cas9 endonucleases reveal RNA-mediated conformational activation

Martin Jinek et al. Science. .

Abstract

Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

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Figures

Fig. 1
Fig. 1. Crystal structure of SpyCas9 reveals an open bilobed architecture and nucleic acid binding clefts
(A) Cartoon schematic of the polypeptide sequence and domain organization for the type II-A Cas9 protein from S. pyogenes (SpyCas9). Cas9 is predicted to contain a single HNH nuclease domain and a single RuvC nuclease domain. The RuvC domain is made up of three discontinuous segments (RuvC-I to RuvC-III), with the α-helical lobe inserted between the first and the second segments, and the HNH domain inserted between the second and the third segments. Arg, arginine-rich region; Topo, Topo-homology domain; CTD, C-terminal domain. (B) Orthogonal views of the overall structure of SpyCas9 shown in ribbon representation. Individual Cas9 domains are colored according to the scheme in (A). SpyCas9 consists of a nuclease domain lobe and an α-helical lobe. Disordered segments of the polypeptide chain are denoted with dotted lines. (C) Surface representation of SpyCas9 depicting the two nucleic acid binding clefts on the molecular surface. (D) Surface electrostatic potential map of SpyCas9 colored from −10 kT/e (red) to +10 kT/e (blue) (61). (E) Surface representation of SpyCas9 colored according to evolutionary conservation. The representation was generated using the ConSurf server (62) based on the multiple sequence alignment of type II-A Cas9 proteins shown in fig. S1. A disordered segment (residues 571Spy to 586Spy, indicated with a black dashed line) covers the apparently conserved patch on the reverse convex surface of SpyCas9.
Fig. 2
Fig. 2. Cross-linking identifies a PAM binding region adjacent to the active-site cleft
(A) Model of noncomplementary DNA strand bound to the RuvC domain based on a superposition with the DNA-bound complex of Thermus thermophilus RuvC Holliday junction resolvase [Protein Data Bank (PDB) entry 4LD0]. The modeled DNA strand contains three nucleotides upstream and three nucleotides downstream of the scissile phosphate. Divalent ions in the RuvC active site are depicted as pink spheres. (B) Zoomed-in view of the RuvC cleft showing the modeled nontarget DNA strand (stick format, scissile phosphate indicated with yellow arrowhead) and the predicted path of the downstream (3′) sequence containing the PAM (orange ball and string). Disordered loops identified by cross-linking are denoted with dashed lines. (C) Cartoon (left) showing the design and workflow of cross-linking experiments with DNA substrates containing BrdU nucleotides for LC-MS/MS analysis. The guide/target sequence is depicted in red, and the PAM is highlighted in yellow. The denaturing polyacrylamide gel (right) demonstrates the generation of covalent peptide-DNA adducts with catalytically inactive SpyCas9 (dCas9) after UV irradiation and trypsin digestion. (D) DNA cleavage activity assays with SpyCas9 constructs containing mutations in residues identified by cross-linking and LC-MS/MS experiments. The asterisk denotes trimming of the nontarget strand.
Fig. 3
Fig. 3. Crystal structure of AnaCas9 defines the conserved structural core of Cas9 enzymes
(A) Cartoon schematic of the polypeptide sequence and domain organization for the type II-C Cas9 protein from A. naeslundii (AnaCas9). (B) Overall structure of AnaCas9 shown in ribbon representation. Individual Cas9 domains are colored according to the scheme in (A). A disordered segment connecting a RuvC motif and Arg-rich region is denoted with a dashed line. The disordered region in the helical lobe is denoted with a dotted line box. A green sphere denotes a bound zinc ion in the HNH domain. (C) Superposition of AnaCas9 [colored as in (A)] with SpyCas9 (colored light orange). (D) Close-up view of the active site of AnaCas9 HNH domain (yellow) superimposed with the structure of I-HmuI–DNA complex (PDB entry 1U3E). The DNA cleavage product in the I-HmuI–DNA complex is colored orange, and I-HmuI and its bound Mn2+ ion are colored gray. (E) Close-up view of the AnaCas9 RuvC active site (marine, bound Mn2+ ions shown as purple spheres) overlaid with the structure of RNase H and its bound Mn2+ ions (gray) complexed with a DNA-RNA duplex (orange) (PDB entry 3O3H). (F) Surface representations of SpyCas9 (left panel) and AnaCas9 (right panel) with conserved RuvC, HNH, Arg-rich, Topo-homology, and the conserved cores of the C-terminal domains, colored as in Fig. 1A. The structurally preserved portion of the α-helical lobe is colored green. The nonconserved regions of each protein are colored in gray.
Fig. 4
Fig. 4. Both SpyCas9 and AnaCas9 adopt autoinhibited conformations in the apo state
(A) Models of substrate binding by the HNH domains in SpyCas9 (left) and AnaCas9 (right), based on the superposition of the Cas9 structures with the product-bound complex of the homing endonuclease I-HmuI (PDB entry 1U3E). A 17-bp B-form DNA duplex that covers 3-bp 5′ and 14-bp 3′ of the scissile phosphate is shown. The Cas9 proteins are shown in the same orientation, based on superposition of the respective HNH domains. The HNH domains are depicted in yellow, the RuvC domains are depicted in blue, and residues 1049Spy to 1059Spy of the RuvC domain are shown in black. (B) Zoomed-in view of the HNH domain (yellow) active site in SpyCas9 occluded by the 1049Spy to 1059Spy β-hairpin (black).
Fig. 5
Fig. 5. RNA loading positions the two major lobes of SpyCas9 around a central channel
(A to C) Single-particle EM reconstructions of negatively stained apo-SpyCas9 (A), SpyCas9:RNA:DNA (B), and SpyCas9:RNA (C) at 19-, 19-, and 21-Å resolution (using the 0.5 FSC criterion), respectively. Cartoon representations of the structures are shown (left). The structures are aligned on the basis of the optimal CCCs between the independent α-helical lobes (gray). The smaller RuvC lobe (blue) in SpyCas9:RNA:DNA and SpyCas9:RNA rotates by ~100° [arrow in (B)] with respect to this lobe in the apo-Cas9 structure (transparent mesh) to form a central channel (black dashed line). There is a ~50° rotation [arrow in (C)] of the smaller lobe of SpyCas9:RNA along an axis perpendicular to this channel relative to SpyCas9:RNA:DNA.
Fig. 6
Fig. 6. Bound target DNA and guide RNAs span the central channel
(A and B) Biotinylated DNA duplexes were labeled with streptavidin (SA) at either the end distal to the PAM (A, non-PAM) or both ends (B). From left to right: schematic of structures and labels, three representative reference-free 2D class averages, the corresponding reference-free 2D class average of unlabeled SpyCas9:RNA:DNA, a 2D difference map between the unlabeled and labeled structures, and the corresponding reprojection of the SpyCas9:RNA:DNA structure. The SpyCas9:RNA:DNA reconstruction is shown on the right with superimposed 3D difference density at ≥5σ (green) between the SpyCas9:RNA:DNA reconstruction and the SA-labeled reconstruction. (C and D) Single-particle EM analyses of SpyCas9:RNA labeled with SA at the 3′ end of the crRNA (C) or tracrRNA (D). Data are shown as in (A), with the 3D difference density at ≥6σ depicted in orange. The width of the boxes is ~316 Å.
Fig. 7
Fig. 7. SpyCas9 wraps around target DNA
(A) The central channel of the SpyCas9:RNA:DNA reconstruction (transparent surface) can easily accommodate ~25 bp of an A-form duplex (red). (B) Footprinting experiment with target DNA bound by SpyCas9:RNA. A 55-bp DNA substrate was 5′-radiolabeled on either the target or the nontarget strand and incubated with catalytically inactive SpyCas9:RNA programmed with a complementary crRNA (targeting) or a mismatched control crRNA (nontargeting), before being subjected to exonuclease III (left) or nuclease P1 (right) treatment. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis; markers generated via digestion with Bgl I and Fok I restriction enzymes and wild-type SpyCas9:RNA are labeled. The borders of the DNA target protected by SpyCas9:RNA are indicated in red next to the gel and with a gray box (bottom), and nucleotides susceptible to P1 digestion are indicated in red next to the gel and with hashtags in the schematic at the bottom.
Fig. 8
Fig. 8. Model for RNA-induced conversion of Cas9 into a structurally activated DNA surveillance complex
Upon binding the crRNA:tracrRNA guide, the two structural lobes of Cas9 reorient such that the two nucleic acid binding clefts face each other. This generates a central DNA binding channel, which allows access to dsDNA. Target DNA binding in the central channel and PAM-dependent R-loop formation result in a further structural rearrangement. Here, the nuclease domain lobe undergoes further rotation relative to the α-helical lobe, fully enclosing the DNA target, and the two nuclease domains engage both DNA strands for cleavage.
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