The structural biology of CRISPR-Cas systems

Abstract

Prokaryotic CRISPR-Cas genomic loci encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. Acquired and heritable immunity against bacteriophage and plasmids begins with integration of ∼30 base pair foreign DNA sequences into the host genome. CRISPR-derived transcripts assemble with CRISPR-associated (Cas) proteins to target complementary nucleic acids for degradation. Here we review recent advances in the structural biology of these targeting complexes, with a focus on structural studies of the multisubunit Type I CRISPR RNA-guided surveillance and the Cas9 DNA endonuclease found in Type II CRISPR-Cas systems. These complexes have distinct structures that are each capable of site-specific double-stranded DNA binding and local helix unwinding.

Fig. 1. Overview of CRISPR-Cas adaptive immune systems

A typical CRISPR locus consists of an AT-rich leader sequence upstream of the CRISPR array (gray box), a series of repetitive DNA sequences called repeats (brown diamond) and non-repetitive spacers (colored boxes), as well as a set of CRISPR-associated (cas) genes (shown as arrows). In the acquisition phage, new spacer (highlighted in green) derived from the viral DNA or other invasive genetic elements is excised and inserted into the CRISPR array by an undetermined mechanism. During the expression stage, cas genes encoding a diverse set of Cas proteins are transcribed and translated, and the CRISPR array is transcribed into a long precursor CRISPR RNA (pre-crRNA). In Type I and III systems, pre-crRNA is further cleaved into small mature crRNAs by Cas6 endoribonucleases, whereas in Type II systems this processing involves a small trans-activating crRNA (tracrRNA) to form base pairs with the repeat sequence of the pre-crRNA transcript, followed by host RNase III cleavage within the repeat region. In the interference stage, mature crRNAs serves as guides to direct Cas proteins, either as an assembled large multisubunit surveillance complex in Type I & III systems or a single multifunctional Cas9 protein in Type II system, to the invading nucleic acids containing the matching sequence and PAM motif for degradation. Notably, PAM is not required in Type III CRISPR-Cas system.

Fig. 2. X-ray crystal structures of Type I-E Cascade surveillance complex in the pre-target-bound state and ssDNA-bound state

(a) Schematic of the Type I-E CRISPR-Cas operon. (b) Two structures of the pre-target-bound Cascade-crRNA complex projected in the same orientation (left, PDB ID: 1VY8; right, PDB ID: 4U7U). (c) Overall structure of the Cascade-crRNA-ssDNA complex (left, PDB ID: 4QYZ) and structure superimposition of the pre-target-bound state and ssDNA target-bound state (right). For clarity, the Cascade in IVY8 is highlighted in orange, while it is colored teal in 4QYZ. The backbone of crRNA and bound ssDNA is depicted as black tube and purple tube, respectively. (d) Six prominent β-hairpin elements act as “wedges” to make kinks in the RNA at 6-nt intervals in the pre-target-bound (left) and ssDNA target-bound (right) conformations. (e) Left: stereo view of L2 loop (showing in the dotted circles)-containing helical bundle in the ssDNA target-bound (teal) and pre-target-bound (orange) conformations, highlighting the structural change of this loop upon binding to ssDNA target (purple). Right: Close-up view of the base-specific interactions between the Cse1 L1 loop in the pre-target-bound state (orange) and the AAC triplet of 5’-handle crRNA (colored black).

Fig. 3. EM structures of the Type I-E Cascade surveillance complex in the pre-target-bound state and target-bound state (same view as Fig. 2)

(a) Left: The 8-Å cryo-EM pre-target-bound structure of Cascade-crRNA complex (EMD-5314, colored by domain as shown in Fig. 2c). Right: Docking of the crystal structure of Cascade-crRNA-ssDNA complex (PDB ID: 4QYZ) into the pre-target-bound EM map (EMD-5314, colored light blue) showing ssDNA strand traveling along the crRNA backbone. (b) Left: cryo-EM reconstruction of dsDNA-bound Cascade at 9-Å resolution (EMD-5929, colored as in Fig. 3a). The density corresponding to the PAM-proximal dsDNA end is shown in light pink. Middle: The 20-Å negative-stain EM reconstruction of Cas3-bound dsDNA-Cascade complex (EMD-5930, colored as Fig. 3a). Left: Docking the crystal structure of Cascade-crRNA-ssDNA complex into the Cas3-bound dsDNA-Cascade EM density map (light pink) reveals that the additional densities correspond to the PAM-proximal dsDNA end and Cas3.

Fig. 4. X-ray crystal structures of Type II CRISPR-Cas9 proteins in the apo state

(a) A typical structure of CRISPR locus in Type II CRISPR-Cas system. The signature Cas protein, Cas9, is colored green; while the unique regulatory RNA, tracrRNA, is shown as an orange box. (b) Schematic diagrams showing the domain organization of the Type II-A Cas9 protein from S. pyogenes (SpyCas9, PDB ID: 4CMP) and the Type II-C Cas9 protein from A. naeslundii (AnaCas9, PDB ID: 4OGE). Amino acid residue numbers at the domain boundaries are indicated. (c) Orthogonal views of the overall structures of SpyCas9 (left) and AnaCas9 (right) shown in ribbon representation. Individual Cas9 domains are colored according to the scheme in (b) and locations of the HNH and RuvC active sites are indicated by arrows.

Fig. 5. X-ray crystal structures of Type II CRISPR-Cas9 in the target-bound states

(a) Left: Ribbon representation of the SpyCas9-sgRNA-ssDNA ternary complex (PDB ID: 4OO8). SpyCas9 domains are colored as in Fig. 4b, unless otherwise stated. Two yellow stars represent RuvC and HNH active sites, respectively. Middle: Stereo view of the apo structure of SpyCas9 (PDB ID: 4CMP), after superposition onto the structure of SpyCas9-sgRNA-ssDNA ternary complex (not shown for clarity). Right: Structure of the sgRNA:ssDNA heteroduplex. The Arg-rich region in the bridge helix is highlighted in purple. (b) Left: Ribbon representation of the SpyCas9 in complex with sgRNA and a partially duplexed target DNA containing a 5’-TGG-3’ PAM sequence (PDB ID: 4UN3), viewed in the same orientation as in Fig. 5a after optimal superimposition. Middle: Ribbon diagram showing the structure of the sgRNA:partial duplex DNA complex. Right: Close-up view of PAM binding region and +1 phosphate (+1 P, orange sphere). The 5’-NGG-3’ trinucleotide in the non-target strand DNA (purple) is highlighted with blue with stars. The black arrow denotes the directionality of 5’ end of target strand ssDNA.