Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

Abstract

Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.

Inactivation of Target RNA Cleavage of a III-B CRISPR-Cas System Induces Robust Autoimmunity in Saccharolobus islandicus

“…When tgRNA cleavage activity was discovered in 2009, it was proposed that the activity could silence targeted viral genes to block the viral life cycle [ 15 ]. Indeed, the backbone cleavage can reduce the mRNA level of target genes in vivo and result in loss of function of a number of targeted genes in Sulfolobales [ 7 , 58 , 69 , 74 , 75 , 76 ]. In addition, the S. thermophilus Csm can prevent infection of E. coli cells by an ssRNA virus [ 17 ].…”

“…Upon the first invasion of a virus, the system recognizes a DNA sequence from its genome, and cleaves and inserts the DNA sequence into a CRISPR array as the first new spacer. Then, the system produces mature CRISPR RNA (crRNA) from transcripts of the CRISPR array, and in the interference stage, the crRNA and Cas proteins form ribonucleoprotein (RNP) complexes that restrict the re-occurring virus, specifically in an RNA-guided fashion [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. CRISPR-Cas systems fall into two classes and at least six different types.…”

Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems

“…The most widespread type-I and type-III class 1 systems co-exist in many prokaryotic genomes (30)(31)(32). Such a co-existence is well represented in genomes from a crenarchaeal order Sulfolobales, two-thirds of which harbor both the systems (4,33,34). For example, Sulfolobus islandicus encodes two CRISPR loci, one subtype I-A adaptation and two subtype III-B interference modules (34).…”

“…Recognition of a PAM and a crRNA complementary sequence in the target DNA by the type-I interference complex (known as CRISPR-associated complex for antiviral defense or Cascade) recruits the Cas3 endonuclease for degradation of the non-target strand (Fig 1B) (38,39). By contrast, the type-III interference (cmr/csm) complexes recognize a newly-transcribed phage RNA in a PAM-independent fashion by base pairing with a seed motif at the 3' -end of the crRNA (4,(30)(31)(32)(33)(34). Self-targeting during type-III interference is prevented by a mismatch at the 5' -end of the crRNA (Fig 1A) (40,41).…”

Cyclic Tetra-Adenylate (cA4) Recognition by Csa3; Implications for an Integrated Class 1 CRISPR-Cas Immune Response in Saccharolobus solfataricus

“…Relevant to the work described here are subtypes I-A, III-B and III-D belonging to class 1 systems. The Saccharolobus solfataricus P2 genome (formerly Sulfolobus solfataricus [ 5 ]) encodes three type I-A, three type III-B and one type III-D module, and harbors six CRISPR loci designated CRISPRs A–F ( Figure S1 ) [ 6 , 7 , 8 ]. Type I-A effector complexes, also known as archaeal Cascade (aCascade), target double-stranded DNA in a PAM-dependent manner [ 1 , 9 ], while the type III-B and III-D effector complexes target transcriptionally active DNA and mRNA in a PAM-independent manner.…”