Accessory Nucleases Provide Robust Antiparasite Immunity for Type III CRISPR-Cas Systems

To protect against parasites like bacteriophages and plasmids, bacteria employ diverse and sophisticated defence systems. Clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas systems are adaptive immune systems that can integrate short “spacers” from a parasite into its CRISPR locus as a form of immunological memory. Upon reinfection, short RNAs transcribed from the CRISPR locus can guide Cas proteins to the viral genome through complementary base pairing. Cas nucleases then destroy the invader’s genome. To date, six major types and multiple subtypes of CRISPR systems exist, each with their own signature genes and mechanisms of action. Type III CRISPR systems are uniquely able to destroy both the parasite’s DNA and RNA. Type III loci contain Cas10 and Csm2-5, which make up the main Cas10-Csm targeting complex. In addition, loci typically contain an ancillary RNase, csm6 or csx1. Upon target transcription, the Cas10-Csm complex recognises a viral transcript containing a target, which activates DNase activity of Cas10, leading to the destruction of the invader. In addition, it was recently discovered that the Palm domain of Cas10 can synthesise cyclic oligoadenylate second messengers (cA). cA can activate Csm6 by binding to the latter’s CARF domain. In this work, I first elucidate and illuminate the role and mechanism of action of Csm6 during anti-plasmid immunity in staphylococci. I show that Csm6 is required for efficient immunity against a weakly transcribed target but is dispensable against a welltranscribed target. Moreover, in vivo, Csm6 is a non-specific RNase, targeting both host and invader transcripts. This induces a transient growth arrest in the host cell, which is relieved upon target clearance. This growth arrest “buys time” for the Cas10-Csm complex to eliminate the plasmid, which is required for clearance against weakly transcribed targets. Further, I expand and characterise broader arsenal of cA-activated CARF genes that type III systems use during immunity. I identify Card1, a nuclease that can degrade both ssDNA and ssRNA in vitro. These activities required divalent cations, and were activated by cA4. In Staphylococcus aureus, Card1 induces a growth arrest upon activation, and enhance anti-phage immunity. The protection is most likely primarily through the ssDNase activity, since no RNA degradation was detected in vivo. Together with collaborators, we were also able to solve the crystal structure of apo-, cA4-, and cA6- bound Card1 structures, revealing the conformational changes allowing catalysis upon ligand binding. I also identify TM-1, a transmembrane helix-CARF gene that also causes a growth arrest in S. aureus when stimulated by cA production. The mechanism of TM-1 remains to be elucidated, but likely represents the first CRISPR protection mechanism not mediated by degrading nucleic acid. Altogether, my work both deepens and broadens our understanding of the ligandmediated immune response of type III CRISPR systems. Robust immunity is obtained by coupling specific invader destruction (Cas10 DNase activity) with non-specific host and parasite growth arrest (Csm6/Card1/TM-1). This serves as a broader paradigm of how bacteria can use different catalytic activities and different systems to resist their parasites

Skolefravær – slik elever opplever og erfarer det : En kvalitativ studie om fire ungdommers erfaringer med skolefravær, og deres meninger om hva som skal til for å øke tilstedeværelsen

The subject of this study is school refusal. The purpose is to gain greater insight and knowledge on the topic of school refusal from the students' own experiences, based on the research question: "How do students with school refusal experience their own situation, and what do they think can contribute to increase attendance?".A qualitative method with an inductive design has been used, and data collection has been done using semi-structured interviews. The findings show that school refusal is not experienced as a choice, but as an avoidance of unpleasant situations. The findings also show that young people relate school absence both to conditions within themselves, but also in situations related to school. School refusal started at primary school and gradually increased. A lack of belonging has resulted in a loss of confidenceto make the changes to succeed in attending school every day. Nevertheless,they express a hope and a desire to be able to participate with peers and get an education. The most important person for this to happen is the teacher. They want the teacher to see them, listen to them and be interested in them. Based on this, the teacher can be the one who supports the youth, so that they can gain positive experiences and increase attendance at their own pace. The findings are in line with previous research on school refusal, which emphasizes the importance of the school to facilitate measures that promote attendance. Keywords: Absenteeism, school refusal, truancy, school refusal behavior, self-determination theor