Coupling transcriptional activation of CRISPR–Cas system and DNA repair genes by Csa3a in Sulfolobus islandicus

Abstract

CRISPR–Cas system provides the adaptive immunity against invading genetic elements in prokaryotes. Recently, we demonstrated that Csa3a regulator mediates spacer acquisition in Sulfolobus islandicus by activating the expression of Type I-A adaptation cas genes. However, links between the activation of spacer adaptation and CRISPR transcription/processing, and the requirement for DNA repair genes during spacer acquisition remained poorly understood. Here, we demonstrated that de novo spacer acquisition required Csa1, Cas1, Cas2 and Cas4 proteins of the Sulfolobus Type I-A system. Disruption of genes implicated in crRNA maturation or DNA interference led to a significant accumulation of acquired spacers, mainly derived from host genomic DNA. Transcriptome and proteome analyses showed that Csa3a activated expression of adaptation cas genes, CRISPR RNAs, and DNA repair genes, including herA helicase, nurA nuclease and DNA polymerase II genes. Importantly, Csa3a specifically bound the promoters of the above DNA repair genes, suggesting that they were directly activated by Csa3a for adaptation. The Csa3a regulator also specifically bound to the leader sequence to activate CRISPR transcription in vivo. Our data indicated that the Csa3a regulator couples transcriptional activation of the CRISPR–Cas system and DNA repair genes for spacer adaptation and efficient interference of invading genetic elements.

The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling

“…It should be noted that the type III CRISPR locus of S. solfataricus contains a number of CARF domain proteins and their contribution to immunity has not yet been studied. In particular, the CARF-family putative transcription factor Csa3 appears to be involved in transcriptional regulation of CRISPR loci, including the adaptation and type I-A effector genes, when activated by cA 4 (Liu et al , 2015, Liu et al , 2017). These observations suggest that the cOA signal may transcend type III CRISPR defence in some cell types by activating multiple defence systems.…”

Fidelity of Prespacer Capture and Processing is Governed by the PAM Mediated Interaction of Cas1-2 Adaptation complex in Escherichia coli

“…The existence of such DNA fragments is infinitesimal in the RecBCD products, hinting at the involvement of an additional processing step to generate befitting substrates. Recent studies in Sulfolobus solfataricus (type I-A), Sulfolobus islandicus (type I-A), Bacillus halodurans (type I-C), Synechocystis sp.6803 (type I-D), Pyrococcus furiosus (type I-G) and Geobacter sulfurreducens (type I-U) (Almendros et al, 2019;Kieper et al, 2018;Lee et al, 2018;Liu et al, 2017;Rollie et al, 2018;Shiimori et al, 2018;Zhang et al, 2019) highlighted the indispensable role of Cas4 nuclease in PAM selection and prespacer processing. The occurrence of Cas4 is predominantly limited to type I CRISPR-Cas system with the exception of subtypes I-E and I-F .…”

Cas4 nucleases can effect specific integration of CRISPR spacers

“…Spacer acquisition occurs by two related but differing mechanisms via de novo acquisition and primed acquisition pathways. The former requires Cas1 and Cas2 in the subtype I-E system of E. coli (12, 16–18), Cas1, Cas2, Csa1, and Cas4 in the subtype I-A system of S. islandicus (19), and Cas1, Cas2, Cas9, and Csn2 in S. thermophilus subtype II-A systems (20, 21). In contrast, primed acquisition involves activation by a pre-existing spacer in a CRISPR locus that matches the targeted genetic element and this then triggers spacer acquisition for different Type I CRISPR–Cas systems (15–17, 22, 23).…”

“…For example, most spacers (95%) in subtype I-E CRISPR arrays of E. coli are 32 bp (24) and that is likely to reflect the structure of the substrate-binding Cas1-Cas2 complex (25). But for other systems, including those of subtypes I–A and II–A (26, 27) spacer lengths vary and, moreover, they require proteins additional to Cas1 and Cas2 for specific adaptation (19–21). Most of these additional proteins belong to the Cas4 family and carry nuclease activities.…”

“…Most of these additional proteins belong to the Cas4 family and carry nuclease activities. Recently, we showed that two Cas4 family proteins, Cas4 and Csa1, are essential for CRISPR adaptation in S. islandicus (19) and more recent studies have shown that the Cas4 nucleases can facilitate PAM recognition and influence spacer lengths and adaptation efficiency (28–30). In particular, the two Cas4 proteins (Cas4-1 and Cas4-2) in Pyrococcus furiosus subtype I-A system have been observed distinct activities: Cas4-1 specifies the upstream PAM while Cas4-2 specifies the conserved downstream motif (30).…”