Conformational regulation of CRISPR-associated nucleases

Abstract

Adaptive immune systems in bacteria and archaea rely on small CRISPR-derived RNAs (crRNAs) to guide specialized nucleases to foreign nucleic acids. The activation of these nucleases is controlled by a series of molecular checkpoints that ensure precise cleavage of nucleic acid targets, while minimizing toxic off-target cleavage events. In this review, we highlight recent advances in understanding regulatory mechanisms responsible for controlling the activation of these nucleases and identify emerging regulatory themes conserved across diverse CRISPR systems.

Figure 1. Diverse CRISPR systems defend against DNA- and RNA-based invaders

A. Phylogenetic tree of CRISPR system Types with nucleic acid substrates that are bound and cleaved. DNA targeting nucleases are labeled red and RNA targeting nucleases are labeled blue. Genes coding for multi-subunit complexes are indicated with the brackets. B. Cartoon representations of each CRISPR system targeting DNA phage, RNA phage, or both.

Figure 2. Class 2 Nuclease activation mechanisms

A. Activation of the Type II Cas9 nuclease (grey with red outline) relies on several regulatory checkpoints. Cas9 adopts a bi-lobed structure that consists of a nuclease-lobe (Nuc lobe) and an alpha-helical recognition lobe (Rec lobe). Single-guide-RNA loading causes a conformational rearrangement in the Rec lobe. To bind duplex DNA, a PAM motif (dark red) is recognized followed by 3′ seed complementation. Directional unwinding of the duplex to the PAM-distal side of the guide induces a conformational rearrangement of two peptide linkers (L1 and L2 colored blue and orange) that stabilize the active conformation of the HNH and RuvC nuclease domains (colored red) to make a blunt-end cut in duplex DNA. B. Type V nucleases (Cas12a, Cas12b, and Cas12c; also known as Cpf1, C2c1, and C2c3) also adopt a bi-lobed architecture, are loaded with an RNA-guide and use PAM and seed recognition to initiate target binding with DNA. However, the DNA cleavage mechanisms may involve the RuvC domain and Nuc domain, or just the RuvC domain. C. Like Type II and V, the Type VI (Cas13, also known as C2c2) proteins adopt a bilobed architecture and are programmed with an RNA-guide. Type VI nucleases bind RNA with a central seed within the RNA-guide, and an adjacent sequence called the PFS (Protospacer Flanking Site) plays a role in nuclease activation.

Figure 3. Regulation of Class 1 Cas nucleases

A. Type I-E Cascade binds to both interference (red) and priming (orange) DNA targets. Binding of dsDNA causes a conformational change in the Cas8e and Cse2 subunits (blue arrows). When bound to interference targets the Cas8e subunit adopts a closed conformation that recruits nuclease active Cas3 (red). When bound to priming targets the Cas8e subunit adopts an open conformation that does not recruit Cas3 directly, but relies on the presence of Cas1–Cas2 for the recruitment of nuclease repressed Cas3 (gray). Cas3 helicase activity produces looped sections of ssDNA which can act as sources of new spacers acquired during priming. B. In Type III systems the multi-subunit complexes bind ssRNA complementary to the crRNA-guide and RNase active sites in the Cas7 backbone subunits (blue) cleave the RNA at regular 6-nt intervals. In addition, RNA binding also activates non-specific DNase activity of the Cas10 subunit, which in some systems also requires recognition of the 3′-end flanking sequence (red). Cleavage and release of the RNA deactivates the Cas10 subunit (gray) and ensures temporal control.