Unity among the diverse RNA-guided CRISPR-Cas interference mechanisms

Abstract

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are adaptive immune systems that protect bacteria and archaea from invading mobile genetic elements (MGEs). The Cas protein-CRISPR RNA (crRNA) complex uses complementarity of the crRNA "guide" region to specifically recognize the invader genome. CRISPR effectors that perform targeted destruction of the foreign genome have emerged independently as multi-subunit protein complexes (Class 1 systems) and as single multi-domain proteins (Class 2). These different CRISPR-Cas systems can cleave RNA, DNA, and protein in an RNA-guided manner to eliminate the invader, and in some cases, they initiate programmed cell death/dormancy. The versatile mechanisms of the different CRISPR-Cas systems to target and destroy nucleic acids have been adapted to develop various programmable-RNA-guided tools and have revolutionized the development of fast, accurate, and accessible genomic applications. In this review, we present the structure and interference mechanisms of different CRISPR-Cas systems and an analysis of their unified features. The three types of Class 1 systems (I, III, and IV) have a conserved right-handed helical filamentous structure that provides a backbone for sequence-specific targeting while using unique proteins with distinct mechanisms to destroy the invader. Similarly, all three Class 2 types (II, V, and VI) have a bilobed architecture that binds the RNA-DNA/RNA hybrid and uses different nuclease domains to cleave invading MGEs. Additionally, we highlight the mechanistic similarities of CRISPR-Cas enzymes with other RNA-cleaving enzymes and briefly present the evolutionary routes of the different CRISPR-Cas systems.

Keywords: CRISPR-cas; Cas10; Cas12; Cas13; Cas3; Cas9; CasDinG; Cascade; RNA binding protein; crRNA.

Figure 1

Schematic representation of a typical CRISPR-Cas locus (top) and the overall interference complex organization (bottom) for each type belonging to the Class 1 system. The CRISPR array with repeats and spacers is shown in crimson and red. The target strand (TS) of the nucleic acid is in blue, the non-target strand (NTS) is in cyan, crRNA is in red/crimson and the recognition motif (PAM/PFS) is in dark purple for all the systems. A, for the type I system (subtype I-E is shown here), Cas6 bound to the 3′ stem-loop of crRNA is represented in dotted outline since it is not part of the helical filament for certain subtypes (I-A, I-B, and I-D). Cas3 nuclease is a part of the surveillance complex before DNA recognition in some subtypes whereas in others it is recruited after R-loop formation. B, the type III systems (subtype III-A complex is shown here) are broadly categorized into III-A (Csm) and III-B (Cmr) subtypes. In the example depicted, a type III surveillance complex binds to the nascent RNA (target) produced by the RNA polymerase, near the transcription elongation complex (TEC). After binding to a non-self RNA target, Cas10 present in the complex becomes activated for ssDNA cleavage by the HD domain and cOA production by the Palm domains. The cA_n-dependent activation of ancillary proteins initiates non-specific cleavage. C, the organization of the type IV-A surveillance complex on a mature crRNA recruit CasDinG for ATP-dependent DNA unwinding. In all the types, the periodically flipped out crRNA nt can be seen near the thumb domain.

Figure 2

Schematic representation of a typical CRISPR-Cas locus (top) and the organization of the interference complex (bottom) for each type belonging to the Class 2 system. The color scheme is as follows: the effector protein in mint green, crRNA in red/crimson, tracrRNA in orange, TS in blue, NTS DNA in cyan, and the recognition motif (PAM/PFS) in dark purple. Dashed outlines show dispensable genes. Black triangles represent cleavage sites. A, the bilobed type-II Cas9 nuclease (subtype II-A is shown here) assembles with the crRNA and the tracrRNA, followed by targeting and cleavage of dsDNA. B, the type-V Cas12 nuclease (subtype V-A is shown here) binds to the crRNA and cleaves NTS and TS DNA sequentially. C, the ShCAST DNA transposition complex utilizes a catalytically inactive Cas12k for crRNA-tracrRNA dependent and PAM-specific DNA targeting and transposition. Other components such as TnsB, TniQ, and the ribosomal protein S15 are essential for effective transposition. D, the type VI Cas13 nuclease (subtype VI-A is shown here) bound to the crRNA cleaves ssRNA. The dashed region of the target RNA represents the variations in its length and structure.

Figure 3

Structures of the Class 1 interference complexes. A single subunit of Cas5 and multiple subunits of Cas7 form the major filament in all Class 1 interference complexes. A, in type I-E Cascade from Escherichia coli (PDB ID 6C66) (126), the 3′-stem-loop of the crRNA is capped by Cas6 and the 5′-handle is capped by Cas5. The minor filament is composed of Cas11s and Cas8. Cas3 loads onto a bulge in the NTS through its interactions with Cas8. B, the type III-A Csm complex from Thermococcus onnurineus (PDB ID 6MUS) (108) is shown. The minor filament is composed of Cas11s and a large Cas10 subunit. C, the type IV-A system from Pseudomonas aeruginosa (PDB ID 7XG3) (98) has a helical structure similar to other Class 1 systems but lacks the minor filament. In this particular system, Cas5 covers the 5′-repeat of the crRNA and its 3′-stem-loop is capped with Cas6. CasDinG is shown bound to the nicked NTS. Figures were prepared using PyMol (369).

Figure 4

Structure of Class 2 interference complex (top) and the movement of the protein domains upon binding of the surveillance complex to the target nucleic acid (bottom). The lines represent the magnitude of movement of the alpha carbon for each amino acid, and the arrows indicate the direction of movement of each domain from surveillance to the interference state. A, structure of the SpCas9 interference complex (PDB ID 7Z4J) (370). When SpCas9 surveillance complex (PDB ID 4ZT0) (146) transitions to the interference complex (PDB ID 7Z4J), conformational changes occur in the REC and NUC lobes. B, structure of FnCas12a interference complex (PDB ID 6I1K) (236). As the FnCas12a surveillance complex (PDB ID 5NG6) (154) transitions to the interference complex (PDB ID 6I1K), major conformational changes occur in the REC lobe and minor changes occur in the NUC lobe. A part of the NTS (cyan) is not visible in this structure. C, structure of Cas13a interference complex (PDB ID 5XWP) (157). Transitioning from the surveillance complex (PDB ID 5XWY) (157) to the interference complex (PDB ID 5XWP), results in major conformational changes in the NUC lobe. Within each panel, the middle scheme shows the amino acid range for each domain and the structure is color-coded based on this scheme. Figures were prepared using PyMol (369).

Figure 5

A close-up view of the active site of selected Class I Cas proteins. Red asterisks show catalytic residues. A, the active site of Cas3 HD nuclease domain from a type I-E system with two Fe²⁺ ions and the conserved His and Asp residues are shown (PBD ID 4QQW) (164). B, the thumb region of Cas7 in type III systems inserts into the crRNA-target RNA duplex at every sixth nucleotide, positioning a conserved Asp in the catalytic loop near the scissile phosphate, enabling a periodic cleavage in the RNA backbone (PDB ID 3X1L, type III-B) (171). Black triangles indicate the cleavage sites. C, the active site of the Cas10 HD domain belonging to a type III-B system with two transition metal-ions (Mn²⁺) and the conserved His and Asp residues are shown (PDB ID 4W8Y) (371). D, the HEPN active site of Csm6 is organized at the dimer interface (PDB ID 5FSH, type III-A) (372). The Ni²⁺ ion captured by the conserved catalytic triad was proposed to be an artifact from crystallization. E, the type IV-A CasDinG with the important residues of the ATP binding pocket marked with an asterisk (PDB ID 7XF0) (98). We positioned NTS-DNA into 7XF0 based on its position in the CasDinG-ssDNA complex (PDB ID 6FWR) (373). Figures were prepared using ChimeraX (374).

Figure 6

A close-up view of the active site of Class 2 Cas nucleases.Red asterisks show catalytic residues. A, HNH domain of Cas9 (PDB ID 7Z4J, type II-A) (370) consists of a ββα motif and cleaves the TS DNA by a one-divalent metal-ion-mediated catalysis. Red and black triangles, respectively, show 3′hydroxyl and 5′-phosphate ends after TS cleavage. B, RuvC domain of Cas9 (PDB ID 7Z4J, type II-A) cleaves NTS DNA by a two-divalent metal-ion-mediated catalysis. Only part of the NTS is visible since it is disordered in the structure. C, Cas12a (PDB ID 6I1K, type V-A) (236) uses a RuvC active site to cleave both NTS and TS of the DNA. RuvC active site is blocked by a “lid” (pink), which transforms into an α helix (PDB ID 6GTG) (237) upon DNA binding, providing access for ssDNA to reach the catalytic site (divalent metal-ions and the rest of the NTS DNA were not captured in both the Cas12a structures). D, the conserved catalytic residues (RX₄H) of Cas13a′s (PDB ID 5WLH, type VI-A) (295) HEPN1 and HEPN2 domains are positioned for a divalent metal-independent RNA cleavage. RNA was not captured in the structure. Figures were prepared using ChimeraX (374).