And… cut! - how conformational regulation of CRISPR-Cas effectors directs nuclease activity

Abstract

Controlling the conformation of dynamic protein, RNA and DNA molecules underpins many biological processes, from the activation of enzymes and induction of signalling cascades to cellular replication. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) effectors are enzymes tightly controlled by conformational steps that gate activation of nuclease domains core to their function in bacterial adaptive immunity. These precise conformational checkpoints combined with programmable activation specified by RNA guides have driven the success of CRISPR-Cas tools in biotechnology, medicine and beyond. To illustrate the importance of conformation in controlling CRISPR-Cas activity, we review the discrete conformational checkpoints at play in class 2 CRISPR-Cas systems. Using Cas9, Cas12a and Cas13a as examples, we describe how protein and nucleic acid conformations precisely control the loading of guide RNA, the selection of target nucleic acids and the activation of nuclease domains. Much like a director controls the timing of transitions between scenes in a movie, CRISPR effectors use conformational checkpoints to precisely direct their enzymatic activity.

Keywords: CRISPR; RNA; biotechnology; protein conformation.

Figure 1. Conformational checkpoints and molecular mechanisms of Streptococcus pyogenes Cas9.

(A) Apo-SpCas9 poised to bind sgRNA. (PDB: 4CMQ) (B) SpCas9-sgRNA with REC domains rotated from (A) to now bind sgRNA. sgRNA is long enough to interact with REC, PI and RuvC domains. (PDB: 4ZT0) (C) HNH and RuvC nucleases are in inactive conformations in apo-SpCas9 or SpCas9-sgRNA complexes. (D) SpCas9-sgRNA binding target with PAM and 3 bp complementarity to sgRNA spacer. (PDB: 7S38) (E) PAM-interacting residues make specific contacts with the NGG sequence on NTS to facilitate DNA unwinding and to allow sgRNA seed to base pair with the TS. PAM recognition of arginine residues – Arg1333 and Arg1335. (F) REC2 lysine residues interact with the target duplex. REC2 duplex-binding lysine residues – Lys233, Lys234, Lys253 and Lys263. (G) Post-cleavage SpCas9-sgRNA-target with full sgRNA-target complementarity. (PDB: 7S4X) (H) HNH domain of structure in (D) positioned by linkers L1 and L2 to prevent catalysis. (I) HNH domain of structure in (G) positioned by linkers L1 and L2 after complete TS-sgRNA R-loop formation of ~17 bp. L1 residues – 765–780. L2 residues – 906–918. (J) SpCas9 HNH cuts the TS, and RuvC cuts NTS to produce blunt-ended DNA. Active site residues (not including Mg²⁺): RuvC – Asp10, Glu762, His982, His983, Asp986. HNH – Asp837, Asp839, His840, Asn863. PAM, protospacer adjacent motif; PI, PAM-interacting. NTS, non-target strand, TS, target strand. REC, recognition.

Figure 2. Conformational checkpoints and molecular mechanisms of Francisella novicida Cas12a.

RuvC active site residues: Asp917, Glu1006 and Asp1255. Lid-loop residues: 1008–1021. (A) Apo-FnCas12a in elongated conformation. Positively charged surfaces on WED and REC domains recruit gRNA for binding. (PDB: 8H9D). (B) FnCas12a-gRNA, where gRNA binding stabilises the compact conformation of FnCas12a. (PDB: 5NG6). (C) gRNA repeat has a pseudoknot that is bound by the WED and RuvC domains. A nuclease domain cleaves pre-gRNA where it exits the complex at the 5′ end. (D) RuvC is inactived by lid-loop conformation covering active site residues. (E) FnCas12a-gRNA-target binding PAM and beginning to associate with gRNA. (PDB: 6GTC). (F) PAM-interacting lysine residues hold target in place for DNA unwinding to occur and for base pairs to form between gRNA seed and target. This structure exhibits pre-base paired conformation of target. PAM recognition of lysine residues – Lys667, Lys671 and Lys677. (G) FnCas12a-gRNA-target with full target complementarity. REC domains shift to accommodate gRNA-target R-loop. (PDB: 6GTG). (H) The Lid-loop over RuvC active site is opened to switch on catalytic activity. (I) FnCas12a cleaves NTS then TS to produce DNA with a 5′ overhang. (J) FnCas12a-gRNA-target complex in post-cleavage state. The RuvC nuclease remains active and solvent exposed to facilitate non-specific ssDNA cleavage. (PDB: 5MGA).

Figure 3. Conformational checkpoints and molecular mechanisms of Leptotrichia shahii and Leptotrichia buccalis Cas13a.

(A) Apo-LshCas13a with gRNA binding pocket exposed to solvent. (PDB: 5WTJ) (B) LbuCas13a-gRNA ready for target binding. (PDB: 5XWY). (C) A nuclease domain cleaves pre-gRNA where it exits the complex at the 5′ end. (D) LbuCas13a-gRNA-target complex with an activated HEPN. (PDB: 5XWP). (E) HEPN is allosterically activated by interactions with gRNA tag (in the direct repeat) in a kinked conformation. (F) Close proximity of HEPN1 and HEPN2 catalytic residues facilitates nuclease activity. LbuCas13a HEPN active site residues: Arg472, His473, His477, Arg1048, Asn1049 and His1053. Residues 1048 and 1053 have been mutated to Ala. (G) Schematic of active LbuCas13a-gRNA-target cleaving RNA in cis and trans using the same HEPN active site. (H) LshCas13a-gRNA-target with tag:anti-tag pairing. (PDB: 7MDQ). (I) Target containing an anti-tag binds gRNA spacer and the tag. This relaxes the gRNA kink as shown in (E). (J) Tag: anti-tag pairing prevents HEPN catalytic residues from coming into close proximity. LshCas13a HEPN active site residues: Arg597, Asn598, His602, Arg1278, Asn1279 and His1283.

Figure 4. Mechanisms of inhibition by anti-CRISPRs targeting Cas13a, Cas12a and Cas9.

(A) Target binding for Listeria seeligeri Cas13a-gRNA is inhibited by AcrVIA1 by blocking the surface that target RNA is first recognised to prevent duplex formation. (PDB: 6VRB) (B) Target binding for Lachnospiraceae bacterium Cas12a-gRNA is inhibited by AcrVA4 by binding to allosteric switch Arg887, which must rotate towards the interior of the complex to facilitate gRNA-target duplex formation. AcrVA4 locks Arg887 in the inactive conformation to prevent duplex formation. (PDB: 6P7M) (C) HNH conformational shift for Neisseria meningitidis Cas9-sgRNA is inhibited by AcrIIC3 despite duplex formation occurring. 2 copies of NmeCas9-sgRNA-AcrIIC3 (PDB: 6JE9) and NmeCas9-sgRNA-target-AcrIIC3 (PDB: 6JE4) are co-ordinated in a ring-like formation. NmeCas9 HNH domain residues – 541–655.