DNA Motifs and an Accessory CRISPR Factor Determine Cas1 Binding and Integration Activity in Sulfolobus islandicus

Abstract

CRISPR-Cas systems empower prokaryotes with adaptive immunity against invasive mobile genetic elements. At the first step of CRISPR immunity adaptation, short DNA fragments from the invaders are integrated into CRISPR arrays at the leader-proximal end. To date, the mechanism of recognition of the leader-proximal end remains largely unknown. Here, in the Sulfolobus islandicus subtype I-A system, we show that mutations destroying the proximal region reduce CRISPR adaptation in vivo. We identify that a stem-loop structure is present on the leader-proximal end, and we demonstrate that Cas1 preferentially binds the stem-loop structure in vitro. Moreover, we demonstrate that the integrase activity of Cas1 is modulated by interacting with a CRISPR-associated factor Csa3a. When translocated to the CRISPR array, the Csa3a-Cas1 complex is separated by Csa3a binding to the leader-distal motif and Cas1 binding to the leader-proximal end. Mutation at the leader-distal motif reduces CRISPR adaptation efficiency, further confirming the in vivo function of leader-distal motif. Together, our results suggest a general model for binding of Cas1 protein to a leader motif and modulation of integrase activity by an accessory factor.

Multilayered interaction between CRISPR-Cas subtype I-A and adjacently encoded Acrs of virus SIRV2

“…Among the diverse CRISPR-Cas systems, the class 1 CRISPR-Cas, divided into type I, type III and type IV, have a high degree of complexity in terms of their RNA-guided surveillance complex. Subtype I-A CRISPR-Cas systems are widespread in archaea and consist of an interference and spacer acquisition module, both of which have been shown to be active against plasmids and virus infections (1)(2)(3)(4)(5). Notably, it has been shown in Sulfolobales that in response to viral infection the CARF (CRISPR Associated Rossman Fold)/HTH (Helix turn helix)-domain containing regulatory proteins Csa3a (activator of spacer acquisition module) and Csa3b (repressor of the interference module) are transcriptionally regulated to enhance CRISPR-Cas activity (6,7).…”