Required base complementarity between guide RNAs and target sequences in the CRISPR-Cas-based virus defense mechanism of the hyperthermophilic archaeon Sulfolobus solfataricus

Mittels CRISPR/Cas-Systemen erwerben Mikroorganismen Immunität gegen Infektion durch Viren und Eindringen anderer exogener Nukleinsäuren. Der CRISPR/Cas Mechanismus beruht auf der spezifischen Erkennung eindringender Fremd-DNA durch kleine, komplementäre RNAs, die als transkribierte Spacer-Sequenzen aus dem CRISPR locus als CRISPR RNAs (crRNAs) hervorgehen und einen so-genannten Protospacer erkennen, d.h. einen Sequenzabschnitt invasiver Nukleinsäuren, welcher Homologie zu einem Spacer aufweist. Im Gegenzug können Viren durch den Erwerb von Mutationen innerhalb ihrer Protospacer-Sequenz einer möglichen DNA Degradation entkommen und somit die erworbene Resistenz ihres Wirtsorganismus umgehen. Bislang wurden die Auswirkungen von Protospacermutationen auf die CRISPR/Cas-basierende Interferenz vor allem in bakteriellen CRISPR/Cas Systemen untersucht. Um mehr über die Anforderungen für die Interaktion zwischen crRNA und Protospacer während der CRISPR/Cas-Interferenz in Archaea zu lernen, wurde der hyperthermophile Crenarchaeote Sulfolobus solfataricus und sein Virus SSV1 als Modellsystem in dieser Studie verwendet. Für die Arbeit wurden verschiedene Protospacermutanten des rekombinanten Virus SSV1 hergestellt und anschließend in mehreren Transfektionsexperimenten untersucht. Die gentechnisch-veränderten Mutanten trugen eine steigende Anzahl an Mutationen an den jeweiligen Enden der Protospacer-Sequenz; d.h. im Ausmaß von sechs bis 18 aufeinander folgenden Mutationen an beiden Protospacerenden. Trotz der eingeführten sechs Mutationen am „down“-Ende des Protospacers, welches dem 3‘-Ende transkribierter Spacer RNA entspricht, vermittelte das S. solfataricus CRISPR/Cas System beinahe 100% Immunität gegen das rekombinante Virus. Im Gegensatz dazu, wurde die Resistenz gegen das rekombinante Virus stark vermindert (um 75%), als sechs Mutationen am anderen Ende („up“-Ende) des Protospacers eingeführt wurden. Es konnten immer noch 50% CRISPR/Cas-basierende Immunität nachgewiesen werden, als 15 aufeinander folgende Mutationen am Protospacer „down“-Ende vorhanden waren. Ähnlich den erst kürzlich angestellten Beobachtungen in E. coli, erwies sich das CRISPR/Cas System als deutlich toleranter gegenüber Mutationen, die am „down“-Ende des Protospacers auftraten. Dies deutet darauf hin, dass CRISPR/Cas-Interferenz in S. solfataricus durch die Erkennung einer kurzen Sequenz am „up“-Ende des Protospacers initiiert wird. Verglichen mit dem bakteriellen System, schien die erforderliche Basenkomplementarität zwischen Zielsequenz und crRNA während der CRISPR/Cas-Interferenz jedoch sehr gering zu sein.Microbes have evolved various defense strategies in order to prevent viral attack and invasion of foreign DNA, respectively. Clustered regularly interspaced short palindromic repeats (in short: CRISPR) characterize one of those defense mechanisms and are found in many bacterial and most archaeal genomes. CRISPR/Cas systems confer acquired resistance against viruses and plasmids by specifically targeting invasive nucleic acids via sequence complementarity between small spacer-derived CRISPR RNAs (crRNAs) and so called protospacer sequence of the invading (targeted) nucleic acids. Conversely, viruses can find a way to escape CRISPR/Cas resistance by acquisition of mutations within their protospacer sequence. Interestingly, not all protospacer mutations lead to viral escape. So far the impact of protospacer mutations on CRISPR/Cas-mediated interference has mainly been studied in bacterial CRISPR/Cas systems. To learn more about the requirements of crRNA-protospacer interaction during CRISPR/Cas-mediated interference in Archaea, the hyperthermophilic archaeon Sulfolobus solfataricus and its virus SSV1 were used as a model system in this study. Various protospacer mutants of the recombinant SSV1 virus were tested in transfection experiments. The engineered mutants carried increasing numbers of mutations at the two different ends of the protospacer sequence, i.e. six up to 18 consecutive mutations at both ends, respectively. The S. solfataricus CRISPR/Cas system conferred almost 100 % immunity against the recombinant virus containing six mutations at the “down”-end of the protospacer, i.e. at the 3’ end with respect to transcription of the spacer RNA. In contrast, viral resistance was severely reduced (by 75 %) when six mutations were introduced at the other end (“up”-end) of the protospacer. Still 50 % viral resistance was observed when 15 mutations were present at the protospacer “down”-end. Similar to observations recently made in E. coli, the CRISPR/Cas system was significantly more tolerant towards mutations present at the “down”-end of the protospacer than at the other end, suggesting that CRISPR/Cas interference in S. solfataricus might be initiated by the recognition of a short sequence at the “up”-end of the protospacer. In contrast to the bacterial system however, the required base complementarity between the target sequence and crRNA during CRISPR/Cas interference seemed to be very low

Abstracts from the 20th International Symposium on Signal Transduction at the Blood-Brain Barriers

https://deepblue.lib.umich.edu/bitstream/2027.42/138963/1/12987_2017_Article_71.pd

Identification and molecular characterisation of a homozygous missense mutation in the ADAMTS10 gene in a patient with Weill-Marchesani syndrome

Weill-Marchesani syndrome is a rare disorder of the connective tissue. Functional variants in ADAMTS10 are associated with Weill-Marchesani syndrome-1. We identified a homozygous missense mutation, c.41T>A, of the ADAMTS10 gene in a 19-year-old female with typical symptoms of WMS1: proportionate short stature, brachydactyly, joint stiffness, and microspherophakia. The ADAMTS10 missense mutation was analysed in silico, with conflicting results as to its effects on protein function, but it was predicted to affect the leader sequence. Molecular characterisation in HEK293 Ebna cells revealed an intracellular mis-targeting of the ADAMTS10 protein with a reduced concentration of the polypeptide in the endoplasmic reticulum. A large reduction in glycosylation of the cytoplasmic fraction of the mutant ADAMTS10 protein versus the wild-type protein and a lack of secretion of the mutant protein are also evident in our results.In conclusion, we identified a novel missense mutation of the ADAMTS10 gene and confirmed the functional consequences suggested by the in silico analysis by conducting molecular studies

Novel, Innovative Models to Study Ischemia/Reperfusion-Related Redox Damage in Organ Transplantation

The implementation of ex vivo organ machine perfusion (MP) into clinical routine undoubtedly helped to increase the donor pool. It enables not just organ assessment, but potentially regeneration and treatment of marginal organs in the future. During organ procurement, redox-stress triggered ischemia-reperfusion injury (IRI) is inevitable, which in addition to pre-existing damage negatively affects such organs. Ex vivo MP enables to study IRI-associated tissue damage and its underlying mechanisms in a near to physiological setting. However, research using whole organs is limited and associated with high costs. Here, in vitro models well suited for early stage research or for studying particular disease mechanisms come into play. While cell lines convince with simplicity, they do not exert all organ-specific functions. Tissue slice cultures retain the three-dimensional anatomical architecture and cells remain within their naïve tissue-matrix configuration. Organoids may provide an even closer modelling of physiologic organ function and spatial orientation. In this review, we discuss the role of oxidative stress during ex vivo MP and the suitability of currently available in vitro models to further study the underlying mechanisms and to pretest potential treatment strategies

An oligosaccharyltransferase from Leishmania donovani increases the N-glycan occupancy on plant-produced IgG1.

N-Glycosylation of immunoglobulin G1 (IgG1) at the heavy chain Fc domain (Asn297) plays an important role for antibody structure and effector functions. While numerous recombinant IgG1 antibodies have been successfully expressed in plants, they frequently display a considerable amount (up to 50%) of unglycosylated Fc domain. To overcome this limitation, we tested a single-subunit oligosaccharyltransferase from the protozoan Leishmania donovani (LdOST) for its ability to improve IgG1 Fc glycosylation. LdOST fused to a fluorescent protein was transiently expressed in Nicotiana benthamiana and confocal microscopy confirmed the subcellular location at the endoplasmic reticulum. Transient co-expression of LdOST with two different IgG1 antibodies resulted in a significant increase (up to 97%) of Fc glycosylation while leaving the overall N-glycan composition unmodified, as determined by different mass spectrometry approaches. While biochemical and functional features of glycosylation improved antibodies remained unchanged, a slight increase in FcγRIIIa binding and thermal stability was observed. Collectively, our results reveal that LdOST expression is suitable to reduce the heterogeneity of plant-produced antibodies and can contribute to improving their stability and effector functions