CRISPR-Cas Adaptive Immune Systems of the Sulfolobales: Unravelling Their Complexity and Diversity

Abstract

The Sulfolobales have provided good model organisms for studying CRISPR-Cas systems of the crenarchaeal kingdom of the archaea. These organisms are infected by a wide range of exceptional archaea-specific viruses and conjugative plasmids, and their CRISPR-Cas systems generally exhibit extensive structural and functional diversity. They carry large and multiple CRISPR loci and often multiple copies of diverse Type I and Type III interference modules as well as more homogeneous adaptation modules. These acidothermophilic organisms have recently provided seminal insights into both the adaptation process, the diverse modes of interference, and their modes of regulation. The functions of the adaptation and interference modules tend to be loosely coupled and the stringency of the crRNA-DNA sequence matching during DNA interference is relatively low, in contrast to some more streamlined CRISPR-Cas systems of bacteria. Despite this, there is evidence for a complex and differential regulation of expression of the diverse functional modules in response to viral infection. Recent work also supports critical roles for non-core Cas proteins, especially during Type III-directed interference, and this is consistent with these proteins tending to coevolve with core Cas proteins. Various novel aspects of CRISPR-Cas systems of the Sulfolobales are considered including an alternative spacer acquisition mechanism, reversible spacer acquisition, the formation and significance of antisense CRISPR RNAs, and a novel mechanism for avoidance of CRISPR-Cas defense. Finally, questions regarding the basis for the complexity, diversity, and apparent redundancy, of the intracellular CRISPR-Cas systems are discussed.

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

“…Trimming of the 5' end of the crRNA probably occurs by a non-Cas RNase. The absence of Type II systems in archaea is consistent with the absence of RNase-III genes in most archaeal genomes [88]. In the Type II-C system of Neiseria meningitidis, short intermediate crRNA guides are transcribed from multiple promoters embedded within the repeats of the CRISPR array, implying that the system does not require RNase-III [89].…”

RNA-activated DNA cleavage by the Type III-B CRISPR–Cas effector complex

“…Thus, spatial regulation may provide a mechanism for immunity (specific cleavage of transcribing foreign DNA) rather than cell death (cleavage of transcribing host DNA). Targeting of antisense transcripts derived from host CRISPR loci, which have been observed in several species Garrett et al 2015), appears to be prevented by base paring between the 5 ′ handle of the crRNA and the complementary region of the resulting antisense transcript (Supplemental Fig. S6).…”

Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR-Cas interference in Sulfolobus

“…13,35 Possibly, Sulfolobus type I-A interference uses a more primitive system wherein the minor annealing site increases the overall binding specificity.…”

“…[6][7][8][9] These crRNAs assemble with Cas proteins into effector complexes and anneal to spacer-matching sequences (protospacers) on invading genetic elements, or transcripts thereof, which are then cleaved. [10][11][12] Much of the seminal work on crenarchaeal CRISPR-Cas systems has been performed on a few model Sulfolobus species, including Sulfolobus solfataricus P2 and S. islandicus REY15A, which can host a variety of diverse viruses and for which versatile genetic systems have been developed (reviewed in [13][14][15] ). These CRISPR-Cas systems tend to be exceptional in that CRISPR loci are multiple and large, and a mixture of type I-A and different type III systems are generally present.…”

“…These CRISPR-Cas systems tend to be exceptional in that CRISPR loci are multiple and large, and a mixture of type I-A and different type III systems are generally present. 1 Although Sulfolobus CRISPR transcription and processing mechanisms have been elucidated and the modes of CRISPR-Cas adaptation and interference have been studied extensively (reviewed in 13,14 ), questions remain regarding the degree of crRNA-protospacer base pairing stringency required for effective interference. [16][17][18] In initial studies on cRNA-protospacer annealing in the bacteria-specific type II CRISPR-Cas system of Streptococcus thermophilus, effective interference was inferred to require highly stringent base pairing of the crRNA-protospacer sequences.…”