Structural basis of CRISPR-Cas Type III prokaryotic defence systems

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Highlights

  • Type III is the most diverse and complex CRISPR-Cas system.

  • Type III CRISPR-Cas systems cleave RNA and DNA.

  • The effector complexes generate secondary messengers in the form of cyclic oligonucleotides.

  • Secondary messengers activate auxiliary nucleases that complement the immune response.

CRISPR loci and CRISPR-associated (Cas) genes encode an adaptive immune system that protects many bacterial and almost all archaea against invasive genetic elements from bacteriophages and plasmids. Several classes of CRISPR systems have been characterized, of which the type III CRISPR systems exhibit the most unique functions. Members of type III cleave both RNA and DNA not only through their corresponding effector complexes but also by CRISPR-Cas associated proteins activated by second messengers produced by those effector complexes. Furthermore, the recent discovery of second messenger degrading proteins called ring nucleases adds an extra regulatory layer to fine-tune these immunity systems. Here, we review the defense mechanisms that govern type III CRISPR interference immunity systems focusing on the structural information available.

Introduction

In prokaryotes, CRISPR-Cas (clustered regularly interspaced short palindromic repeats and its associated genes) adaptive immune systems provide defense against invasive foreign nucleic acids such as bacteriophages and plasmids [1, 2, 3, 4]. This defense relies on CRISPR arrays, which are short repetitive DNA segments interspersed with unique spacer sequences generated during encounters with invading nucleic acids; hence, constituting a genetic record of previous infections. The CRISPR locus is transcribed and processed to generate mature CRISPR RNAs (crRNAs), which are loaded into Cas protein assembling ribonucleoprotein particles (crRNP), which act as effector complexes that detect and subsequently degrade the invading nucleic acid [5]. Based on the effector complex composition CRISPR-Cas systems are divided in two classes. The class 1 systems (types I, III, and IV) consist of multi-subunit effector complexes, while class 2 systems (types II, V, and VI) comprise single-subunit effectors [6, 7, 8].

Type III is of particular interest as they degrade both RNA and DNA of the invaders [9,10]. Type III is subdivided into subtypes III-A to III-F with different subunits and architectures highlighting the large heterogeneity of this CRISPR-Cas system [8] (Table 1). Type III is characterized by the presence of the multidomain Cas10 signature protein, which is called Csm1 or Cmr2 in Csm/Cmr complexes respectively (Table 2). The cas10 gene product contains an N-terminal HD nuclease domain, two Palm domains, and two small α-helical domains. Subtype III-A/D systems encode the 5-subunit Csm complex (Csm1-Csm5), whereas the subtype III-B/C systems encode the 6-subunit Cmr complex (Cmr1-Cmr6). A distinctive feature of subtype III-C is the apparent inactivation of the Palm domains of the Cas10 protein, while subtype III-D loci typically encode a Cas10 protein that lacks the HD domain [7]. In subtype III-E the csm4 and cas10 genes are absent, while several Cas7 proteins and a putative Csm2-like small subunit (Cas11) are fused (Table 1), thus resembling the large multi-domain proteins of class 2. In subtype III-F, the Cas7, Cas5, and Cas10 subunits show distant but substantial similarity to other type III subtypes, whereas the putative small subunit exhibits no similarity to Cas11. This subtype contains only one Cas7-like protein, and the Palm domains are inactivated [8].

Section snippets

CRISPR-Cas type III immunity mechanism

The type III CRISPR-Cas immunity response is instigated by transcription of a target sequence. The recognition of a target mRNA tethers the complex to the transcription bubble (Figure 1) [9,11, 12, 13], and self-transcripts (Non-cognate Target RNA, NTR) are distinguished from invader derived mRNA transcripts (Cognate Target RNA, CTR) by testing complementarity between a 8-nt repeat-derived sequence of the crRNA, termed 5´-tag, and the corresponding 8-nt of the target RNA, which is termed

Structural basis of immunity by type III CRISPR effector complexes

Among the six subtypes, III-A (Csm) and III-B (Cmr) are the best understood from a structural and functional perspective. The overall architecture of Csm and Cmr crRNP complexes, and their architectural reminiscence to Type I cascade complexes, was revealed in 2013-14 by low-resolution cryo-EM reconstructions together with X-ray crystal structures of subunits and subcomplexes [1,26, 27, 28, 29, 30, 31]. In 2015, a 2.1 Å crystal structure of a chimeric Cmr in complex with a crRNA and an

Structural basis of the ancillary Csm6/Csx1 RNases and their regulatory mechanisms

Cyclic oligoadenylates synthetized by the effector complexes activate RNases from the Csm/Csx families, leading to the degradation of the invasive RNA but also affecting the host (Figure 1). All of them share an overall architecture with an N-terminal cAn-binding CARF (CRISPR-associated Rossmann fold) domain and a C-terminal HEPN (higher eukaryotes and prokaryotes nucleotide-binding) ribonuclease domain, which in some cases are joined by an intermediate HTH (helix-turn-helix) domain. The cAn

Regulating cyclic oligoadenylates signaling

cAn-activated Csm6/Csx1 induce a rather indiscriminate RNA degradation, which could be toxic to the host. Therefore, organisms have developed regulatory strategies to eliminate cAn. As previously mentioned, some members of the family, such as TonCsm6 and EitCsm6, can catalyze cAn degradation in their CARF domains to control the RNase activity [20••,21••,47] (Figure 1). However, there are other strategies to switch-off cAn signaling. For instance, the degradation of cAn molecules has been shown

Conclusion and future perspectives

This review reveals the complexity of Type III CRISPR-Cas systems. The recent structural analyses offered important information of type III-A and III-B effector complexes. However, deciphering the molecular mechanisms of the other subtypes is essential to fully understand the immune response of type III. Although the different subtypes present a common interference pattern, the detailed strategies seem to differ between subtypes. The regulation of the secondary messenger levels and the type of

Conflicts of interest statement

G.M. is a co-founder and Board member of Twelve Bio. The other authors declare no competing interest.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (grant NNF14CC0001). This work was also supported by the cryoEM (grant NNF0024386), cryoNET (grant NNF17SA0030214), and Distinguished Investigator (NNF18OC0055061) grants to GM.

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