Britta Stoll

An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA

Background: CRISPR/Cas systems allow archaea and bacteria to resist invasion by foreign nucleic acids. Results: The CRISPR/Cas system in Haloferax recognized six different PAM sequences that could trigger a defense response. Conclusion: The PAM sequence specificity of the defense response in type I CRISPR systems is more relaxed than previously thought. Significance: The PAM sequence requirements for interference and adaptation appear to differ markedly. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.

Essential requirements for the detection and degradation of invaders by the Haloferax volcanii CRISPR/Cas system I-B

To fend off foreign genetic elements, prokaryotes have developed several defense systems. The most recently discovered defense system, CRISPR/Cas, is sequence-specific, adaptive and heritable. The two central components of this system are the Cas proteins and the CRISPR RNA. The latter consists of repeat sequences that are interspersed with spacer sequences. The CRISPR locus is transcribed into a precursor RNA that is subsequently processed into short crRNAs. CRISPR/Cas systems have been identified in bacteria and archaea, and data show that many variations of this system exist. We analyzed the requirements for a successful defense reaction in the halophilic archaeon Haloferax volcanii. Haloferax encodes a CRISPR/Cas system of the I-B subtype, about which very little is known. Analysis of the mature crRNAs revealed that they contain a spacer as their central element, which is preceded by an eight-nucleotide-long 5′ handle that originates from the upstream repeat. The repeat sequences have the potential to fold into a minimal stem loop. Sequencing of the crRNA population indicated that not all of the spacers that are encoded by the three CRISPR loci are present in the same abundance. By challenging Haloferax with an invader plasmid, we demonstrated that the interaction of the crRNA with the invader DNA requires a 10-nucleotide-long seed sequence. In addition, we found that not all of the crRNAs from the three CRISPR loci are effective at triggering the degradation of invader plasmids. The interference does not seem to be influenced by the copy number of the invader plasmid.

A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii

Background: The Cas6 protein is required for generating crRNAs in CRISPR-Cas I and III systems. Results: The Cas6 protein is necessary for crRNA production but not sufficient for crRNA maintenance in Haloferax. Conclusion: A Cascade-like complex is required in the type I-B system for a stable crRNA population. Significance: The CRISPR-Cas system I-B has a similar Cascade complex like types I-A and I-E. The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1–8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.

Small RNAs for defence and regulation in archaea

Non-coding RNAs are key players in many cellular processes within organisms from all three domains of life. The range and diversity of small RNA functions beyond their involvement in translation and RNA processing was first recognized for eukaryotes and bacteria. Since then, small RNAs were also found to be abundant in archaea. Their functions include the regulation of gene expression and the establishment of immunity against invading mobile genetic elements. This review summarizes our current knowledge about small RNAs used for regulation and defence in archaea.

The immune system of halophilic archaea

Prokaryotes have developed several strategies to defend themselves against foreign genetic elements. One of those defense mechanisms is the recently identified CRISPR/Cas system, which is used by approximately half of all bacterial and almost all archaeal organisms. The CRISPR/Cas system differs from the other defense strategies because it is adaptive, hereditary and it recognizes the invader by a sequence specific mechanism. To identify the invading foreign nucleic acid, a crRNA that matches the invader DNA is required, as well as a short sequence motif called protospacer adjacent motif (PAM). We recently identified the PAM sequences for the halophilic archaeon Haloferax volcanii, and found that several motifs were active in triggering the defense reaction. In contrast, selection of protospacers from the invader seems to be based on fewer PAM sequences, as evidenced by comparative sequence data. This suggests that the selection of protospacers has stricter requirements than the defense reaction. Comparison of CRISPR-repeat sequences carried by sequenced haloarchaea revealed that in more than half of the species, the repeat sequence is conserved and that they have the same CRISPR/Cas type.

Requirements for a successful defence reaction by the CRISPR-Cas subtype I-B system.

Uptake of foreign mobile genetic elements is often detrimental and can result in cell death. For protection against invasion, prokaryotes have developed several defence mechanisms, which take effect at all stages of infection; an example is the recently discovered CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) immune system. This defence system directly degrades invading genetic material and is present in almost all archaea and many bacteria. Current data indicate a large variety of mechanistic molecular approaches. Although almost all archaea carry this defence weapon, only a few archaeal systems have been fully characterized. In the present paper, we summarize the prerequisites for the detection and degradation of invaders in the halophilic archaeon Haloferax volcanii. H. volcanii encodes a subtype I-B CRISPR-Cas system and the defence can be triggered by a plasmid-based invader. Six different target-interference motifs are recognized by the Haloferax defence and a 9-nt non-contiguous seed sequence is essential. The repeat sequence has the potential to fold into a minimal stem-loop structure, which is conserved in haloarchaea and might be recognized by the Cas6 endoribonuclease during the processing of CRISPR loci into mature crRNA (CRISPR RNA). Individual crRNA species were present in very different concentrations according to an RNA-Seq analysis and many were unable to trigger a successful defence reaction. Recognition of the plasmid invader does not depend on its copy number, but instead results indicate a dependency on the type of origin present on the plasmid.

The role of Cas8 in type I CRISPR interference.

We have used genetic and protein biochemical analyses in two archaeal species to identify that Cas8 is essential for CRISPR interference. We provide evidence that Cas8 functions as part of archaeal Cascade, an R-loop forming nucleoprotein complex, recognizing protospacer adjacent motifs (PAMs) on invader DNA. An RNA nuclease activity of Cas8 is also described.

The nuts and bolts of the Haloferax CRISPR-Cas system I-B.

ABSTRACT Invading genetic elements pose a constant threat to prokaryotic survival, requiring an effective defence. Eleven years ago, the arsenal of known defence mechanisms was expanded by the discovery of the CRISPR-Cas system. Although CRISPR-Cas is present in the majority of archaea, research often focuses on bacterial models. Here, we provide a perspective based on insights gained studying CRISPR-Cas system I-B of the archaeon Haloferax volcanii. The system relies on more than 50 different crRNAs, whose stability and maintenance critically depend on the proteins Cas5 and Cas7, which bind the crRNA and form the Cascade complex. The interference machinery requires a seed sequence and can interact with multiple PAM sequences. H. volcanii stands out as the first example of an organism that can tolerate autoimmunity via the CRISPR-Cas system while maintaining a constitutively active system. In addition, the H. volcanii system was successfully developed into a tool for gene regulation.

RNA Structures as Processing Signals

Gene expression is regulated on several levels, one of which is the level of RNA maturation. While we have some knowledge about RNA processing in Bacteria and Eukarya, little is known about this process in the third domain of life, Archaea. The aim of this project is to identify processing signals in the model archaeon, the halophilic archaeon Haloferax volcanii. To achieve that goal, two approaches will be taken: (i) an in silico approach and (ii) an experimental approach. Both approaches are dependent upon and complementary to each other. Processing sites identified in silico will be tested in the laboratory and results from the experimental approach will allow refinement of parameters for the in silico searches. In this interdisciplinary project the partner from computer science/information theory will help designing software tools (based on information theoretic methods) to identify putative positions in the Haloferax genome where tRNA-like structures exist. In addition, the complete set of newly identified processing sites will be searched (and clustered) for common features. The proposed project will lead to the identification of maturation signals in Haloferax volcanii, unravelling the RNA processing pathways and thus this level of regulation of gene expression in Haloferax.