The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers were transcribed. For family I CRISPR loci, the presence of the protospacer CC motif was shown to be important for the occurrence of deletions. The results are consistent with a low level of random dynamic recombination occurring spontaneously, either inter-genomically or intra-genomically, at the repeat regions of Sulfolobus CRISPR loci. Moreover, the relatively high incidence of single-spacer deletions observed for S. islandicus suggests that an additional more directed mechanism operates in this organism.

SummaryRecent studies on CRISPR-based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon Sulfolobus islandicus REY15A encodes three interference modules, one of type IA and two of type IIIB. Earlier we showed that type IA activity eliminated plasmid vectors carrying matching protospacers with specific CCN PAM sequences. Here we demonstrate that interference-mediated by one type IIIB module Cmr-a, and a Csx1 protein, efficiently eliminated plasmid vectors carrying matching protospacers but lacking PAM motifs. Moreover, Cmr-a-mediated interference was dependent on directional transcription of the protospacer, in contrast to the transcription-independent activities of the type IA and type IIIA DNA interference. We infer that the interference mechanism involves transcription-dependent DNA targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S. islandicus REY15A which cooperate functionally by sharing a single Cas6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers.

Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the beta-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were constructed, which genetically complemented DeltapyrEFDeltalacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.

Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic β-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.