Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes

Abstract

The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex via a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site via two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences.

Keywords: active site transformation; allostery; cleavage-ligation equilibrium; inactive-to-active transition; open-closing motions; swiveling motions; twisting motions.

FIGURE 1

Overall structure of a RuvC-catalytically relevant Cas9 complex. (A) Linear structures with color coded domains. (B) Three different orientations of the 5f9r complex with successive rotations of about 70° and 180° along the vertical axis. Two strands of the DNA duplex are in rainbow colors, and gRNA is in grey. Two PAM nucleotides are in large balls-and-sticks. Scissile phosphates for both the tDNA and ntDNA strands are represented by large spheres. (C) Two views of the RuvC-HNH domains. (D) Superposition of the RuvC-HNH domains between the apo-4cmp and the catalytically inactive 5b2r structures. (E) Superposition of the catalytically relevant complexes of 5f9r and 6o0y. (F) Two views of the catalytically inactive and catalytically relevant complexes of 5b2r and 5f9r.

FIGURE 2

Surface representation of RuvC-catalytically relevant complexes in various orientations. Domains are colored as in Figure 1. Missing sidechains and loops were not rebuilt.

FIGURE 3

Two views of HNH domain rotations. (A) Between the RuvC and HNH catalytically relevant complexes of 5f9r and 6o0y. (B) Between the RuvC catalytically relevant and its inactive complexes of 5f9r and 5b2r. See supporting information for animation videos of the domain rotations (Supplementary Video S1).

FIGURE 4

Structures of the RuvC domain in the RuvC catalytically relevant (5f9r) and catalytically inactive (5b2r) complexes. (A) Crystallographic electron density maps for a portion of the 5f9r RuvC structure contoured at 1.5σ. (B) Crystallographic electron density maps for a portion of the 5b2r RuvC structure. (C) Two views of the superposition of part of the two structures. (D,E) A complete view of the entire RuvC domain and zoom-in view of the RuvC catalytic site, with two metal ions computationally modeled. The ntDNA scissile phosphate is shown in magenta and red. (F) Superimposition of the complete RuvC domains of the two structures with the catalytic residues indicated. (G,H) local topological drawings of the RuvC domain in the two structures.

FIGURE 5

HNH catalytic site. (A) A close-up view of the HNH active site in the catalytically relevant complex of 6o0y with one Mg²⁺ ion computationally modeled. (B) A zoomed-out overall view of the entire HNH domain of 6o0y. (C) An overall view of the inactive 5f9r HNH domain. Note that N863 points away from H840. (D) Superposition of the 6o0y and 5f9r HNH domains. (E) Two views of superpositions of the RuvC domain to see relative rotations of the HNH domain between the 5f9r and 6o0y complexes. Rotation axis is indicated by the arrow. (F) Two views of superpositions of the RuvC between the 5f9r and 5b2r complexes.

FIGURE 6

The HNH domain serves as a part of the RuvC active site in the 5f9r structure. (A) The HNH domain is inserted between the β6 stand and α3 helix near the RuvC active site (green side chains plus two modeled Mg ions in gold). The location of K548 (which was built as an alanine residue as well as many other residues also as alanine residues including K510 and some HNH catalytic residues). (B) Two orthogonal views of σ_A-weighted F_o–F_c ED maps retrieved from the PDB contoured at + 1.5σ. (C) Three views of the maps with modeled nucleic acids (which had much stronger ED values).

FIGURE 7

Locations of three MD-derived HNH conformations at the HNH-Rec II interface. (A) HNH (cyan)-Rec (brown) interface from the 5b9r complex. (B) Crystallographic electron density map (contoured at 1.5σ) for the 5f9r complex in two orthogonal views. (C) Alignment of the MD-derived three HNH conformations (gold, blue, and silver) with the 5f9r HNH (cyan) structure. The tDNA scissile phosphate is shown in magenta, Mg²⁺ ion in gold, as well as three catalytic residues (D839/H840 and N863) in an inactive conformation.

FIGURE 8

(A) Two front and back views of the HNH domain from the HNH-activated Cas9 complex of 6o0y. (B) Close-up view of the HNH binding at the minor groove of the RNA/DNA duplex. (C) Three views of the complex superposed with emd-0584 contoured at 6σ (cyan isosurface and salmon isomesh) and 12σ (salmon isosurface).

FIGURE 9

Potential roles of three MD-derived HNH conformations during activation of the HNH active site. (A) Alignment of the three MD-derived conformations of the HNH domain (gold, blue, and silver for conformations 1, 2, and 3, respectively) superimposed onto the 6o0y experimental structure (cyan). Locations of three Lys-to-Ala mutations are shown in large spheres at Cα: K810, red, K848, green, and K855, blue. Metal ion is in medium-size gold sphere. Y836, D861, N863, and H840 residues are shown. (B) Two views of the superposition in the presence of the RNA/DNA duplex. (C) Stereodiagram of a close-up view for showing the relationship of the Y836/D861/N863 three residues (B). (D) A conversion of coiled-coil to α-helix results in a large displacement of N863 (5.4 Å at Cα and 9.5 Å at Oε1). See Supplementary Video S2 for locations of three mutants relative to domain rotations.