Dataset Information

CRISPR-Associated Factor Csa3b Regulates CRISPR Adaptation and Cmr-Mediated RNA Interference in Sulfolobus islandicus.

ABSTRACT: Acquisition of spacers confers the CRISPR-Cas system with the memory to defend against invading mobile genetic elements. We previously reported that the CRISPR-associated factor Csa3a triggers CRISPR adaptation in Sulfolobus islandicus. However, a feedback regulation of CRISPR adaptation remains unclear. Here we show that another CRISPR-associated factor, Csa3b, binds a cyclic oligoadenylate (cOA) analog (5'-CAAAA-3') and mutation at its CARF domain, which reduces the binding affinity. Csa3b also binds the promoter of adaptation cas genes, and the cOA analog enhances their binding probably by allosteric regulation. Deletion of the csa3b gene triggers spacer acquisition from both plasmid and viral DNAs, indicating that Csa3b acted as a repressor for CRISPR adaptation. Moreover, we also find that Csa3b activates the expression of subtype cmr-? and cmr-? genes according to transcriptome data and demonstrate that Csa3b binds the promoters of cmr genes. The deletion of the csa3b gene reduces Cmr-mediated RNA interference activity, indicating that Csa3b acts as a transcriptional activator for Cmr-mediated RNA interference. In summary, our findings reveal a novel pathway for the regulation of CRISPR adaptation and CRISPR-Cmr RNA interference in S. islandicus. Our results also suggest a feedback repression of CRIPSR adaptation by the Csa3b factor and the cOA signal produced by the Cmr complex at the CRISPR interference stage.

SUBMITTER: Ye Q

PROVIDER: S-EPMC7480081 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

CRISPR-Associated Factor Csa3b Regulates CRISPR Adaptation and Cmr-Mediated RNA Interference in <i>Sulfolobus islandicus</i>.

Ye Qing Q Zhao Xueqiao X Liu Jilin J Zeng Zhifeng Z Zhang Zhufeng Z Liu Tao T Li Yingjun Y Han Wenyuan W Peng Nan N

Frontiers in microbiology 20200826

Acquisition of spacers confers the CRISPR-Cas system with the memory to defend against invading mobile genetic elements. We previously reported that the CRISPR-associated factor Csa3a triggers CRISPR adaptation in <i>Sulfolobus islandicus</i>. However, a feedback regulation of CRISPR adaptation remains unclear. Here we show that another CRISPR-associated factor, Csa3b, binds a cyclic oligoadenylate (cOA) analog (5'-CAAAA-3') and mutation at its CARF domain, which reduces the binding affinity. Cs ...[more]

PMID: 32983033

Similar Datasets

Project description:Sulfolobus islandicus is rapidly emerging as a model system for studying the biology and evolution within the TACK lineage of the archaeal domain. As the tree of life grows, identifying the cellular functions of genes within this lineage will have significant impacts on our understanding of the evolution of the last archaeal eukaryote common ancestor (LEACA) and the differentiation of archaea from eukaryotes during the evolution of the modern-day cell. To increase our understanding of this key archaeal organism, we report a novel high-throughput method for targeted gene inactivation in S. islandicus through one-step microhomology-directed homologous recombination (HR). We validated the efficacy of this approach by systematically deleting 21 individual toxin-antitoxin gene pairs and its application to delete chromosomal regions as large as 50 kb. Sequence analysis of 96 ArgD+ transformants showed that S. islandicus can effectively incorporate donor markers as short segments through HR in a continuous or discontinuous manner. We determined that the minimal size of homology allowing native argD marker replacement was as few as 10 bp, whereas argD marker replacement was frequently observed when increasing the size of homology to 30 to 50 bp. The microhomology-mediated gene inactivation system developed here will greatly facilitate isolation of S. islandicus gene deletion strains, making generation of a collection of genome-wide targeted mutants feasible and providing a tool to investigate homologous recombination in this organism.IMPORTANCE Current procedures for the construction of deletion mutants of S. islandicus are still tedious and time-consuming. We developed a novel procedure based on microhomology-mediated HR, allowing for rapid and efficient removal for genetic regions as large as 50 kb. Our work will greatly facilitate functional genomic studies in this promising model organism. Additionally, we developed a quantitative genetic assay to measure HR properties in S. islandicus, providing evidence that the ability to incorporate short, mismatched donor DNA into the genome through HR was probably a common trait for members of the Sulfolobus genus that are recombinogenic.

Dataset Information

CRISPR-Associated Factor Csa3b Regulates CRISPR Adaptation and Cmr-Mediated RNA Interference in Sulfolobus islandicus.

Publications

CRISPR-Associated Factor Csa3b Regulates CRISPR Adaptation and Cmr-Mediated RNA Interference in <i>Sulfolobus islandicus</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets