1311. Simple and reliable in situ CRISPR-Cas9 nuclease visualization tool is ensuring efficient editing in Streptomyces speciesAlen Pšeničnik, Roman Reberšek, Lucija Slemc, Tim Godec, Luka Kranjc, Hrvoje Petković, 2022, original scientific article Abstract: CRISPR-Cas9 technology has emerged as a promising tool for genetic engineering of Streptomyces strains. However, in practice, numerous technical hurdles have yet to be overcome when developing robust editing procedures. Here, we developed an extension of the CRISPR-Cas toolbox, a simple and reliable cas9 monitoring tool with transcriptional fusion of cas9 nuclease to a beta glucuronidase (gusA) visual reporter gene. The Cas9-SD-GusA tool enables in situ identification of cells expressing Cas9 nuclease following the introduction of the plasmid carrying the CRISPR-Cas9 machinery. Remarkably, when the Cas9-SD-GusA system was applied under optimal conditions, 100% of the colonies displaying GusA activity carried the target genotype. In contrast, it was shown that the cas9 sequence had undergone major recombination events in the colonies that did not exhibit GusA activity, giving rise to “escaper colonies” carrying unedited genotype. Our approach allows a simple detection of “escaper” phenotype and serves as an efficient CRISPR-Cas9 optimisation tool.
Keywords: CRISPR-Cas9, gusA visual screening, "CRISPR escaper colonies", Streptomyces
Published in DiRROS: 17.07.2024; Views: 255; Downloads: 201
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1312. CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potatoTjaša Lukan, Florian Veillet, Maja Križnik, Anna Coll Rius, Tjaša Mahkovec Povalej, Karmen Pogačar, Katja Stare, Laura Chauvin, Jean-Eric Chauvin, Kristina Gruden, 2022, original scientific article Abstract: MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci.
Keywords: CRISPR/Cas9, hypersensitive response (HR)-conferred resistance, immune signaling, live cell imaging, Solanum tuberosum (potato), spatiotemporal analysis, stromules, virus resistance
Published in DiRROS: 17.07.2024; Views: 325; Downloads: 222
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1313. Quantitative assessment of the apical and basolateral membrane expression of VEGFR2 and NRP2 in VEGF-A-stimulated cultured human umbilical vein endothelial cellsEsmeralda K. Bosma, Shahan Darwesh, Jia Y. Zheng, Cornelis J. F. van Noorden, Reinier O. Schlingemann, Ingeborg Klaassen, 2022, original scientific article Abstract: Endothelial cells (ECs) form a precisely regulated polarized monolayer in capillary walls. Vascular endothelial growth factor-A (VEGF-A) induces endothelial hyperpermeability, and VEGF-A applied to the basolateral side, but not the apical side, has been shown to be a strong barrier disruptor in blood–retinal barrier ECs. We show here that VEGF-A presented to the basolateral side of human umbilical vein ECs (HUVECs) induces higher permeability than apical stimulation, which is similar to results obtained with bovine retinal ECs. We investigated with immunocytochemistry and confocal imaging the distribution of VEGF receptor-2 (VEGFR2) and neuropilin-2 (NRP2) in perinuclear apical and basolateral membrane domains. Orthogonal z-sections of cultured HUVECs were obtained, and the fluorescence intensity at the apical and basolateral membrane compartments was measured. We found that VEGFR2 and NRP2 are evenly distributed throughout perinuclear apical and basolateral membrane compartments in unstimulated HUVECs grown on Transwell inserts, whereas basolateral VEGF-A stimulation induces a shift toward basolateral VEGFR2 and NRP2 localization. When HUVECs were grown on coverslips, the distribution of VEGFR2 and NRP2 across the perinuclear apical and basolateral membrane domains was different. Our findings demonstrate that HUVECs dynamically regulate VEGFR2 and NRP2 localization on membrane microdomains, depending on growth conditions and the polarity of VEGF-A stimulation.
Keywords: endotelijske celice, EGF, celična biologija
Published in DiRROS: 17.07.2024; Views: 284; Downloads: 196
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1314. A new locality for the blind loach, Eidinemacheilus smithi (Teleostei: Nemacheilidae) in Iranian Zagros : a morpho-molecular approachMohammad Javad Malek-Hosseini, Yaser Fatemi, Hamid Reza Esmaeili, Tjaša Lokovšek, Matjaž Kuntner, 2022, original scientific article Abstract: Four obligate cave fish species have been recorded from Zagros in Iran: Garra typhlops (Bruun and Kaiser, 1944), G. lorestanensis (Mousavi-Sabet and Eagderi, 2016), G. tashanensis (Mousavi-Sabet, Vatandoust, Fatemi and Eagderi, 2016) and Eidinemacheilus smithi (Greenwood, 1976). So far, the only known locality of the latter has been the Loven cave. Here, we extend the known range of the Zagros blind loach (E. smithi) by reporting a new locality, the Tuveh spring, located 31 km south of Loven. We combine morphological evidence with the calculated K2P genetic divergences of 1.39% between Tuveh and Loven to confirm that these populations are conspecific. Our discovery of the second E. smithi population has implications for the conservation of this rare and vulnerable species.
Keywords: fishes, caves, evolution, Irano-Anatolian hotspot
Published in DiRROS: 17.07.2024; Views: 285; Downloads: 192
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1315. Poročilo o preskusu št.: LVG 2024-067 : vzorec št. 2024/00318Nikica Ogris, Špela Hočevar, Zina Devetak, Barbara Piškur, 2024, expertise, arbitration decision Keywords: varstvo gozdov, morfološke analize, program preiskav, Fusarium circinatum, Pinus, borov smolasti rak, PCR
Published in DiRROS: 17.07.2024; Views: 279; Downloads: 0
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1318. Controlling gene expression with deep generative design of regulatory DNAJan Zrimec, Xiaozhi Fu, Azam Sheikh Muhammad, Christos Skrekas, Vykintas Jauniskis, Nora K. Speicher, Christoph S. Börlin, Vilhelm Verendel, Morteza Haghir Chehreghani, Devdatt P. Dubhashi, Verena Siewers, Florian David Fitz, Jens Nielsen, Aleksej Zelezniak, 2022, original scientific article Abstract: Design of de novo synthetic regulatory DNA is a promising avenue to control gene expression in biotechnology and medicine. Using mutagenesis typically requires screening sizable random DNA libraries, which limits the designs to span merely a short section of the promoter and restricts their control of gene expression. Here, we prototype a deep learning strategy based on generative adversarial networks (GAN) by learning directly from genomic and transcriptomic data. Our ExpressionGAN can traverse the entire regulatory sequence-expression landscape in a gene-specific manner, generating regulatory DNA with prespecified target mRNA levels spanning the whole gene regulatory structure including coding and adjacent non-coding regions. Despite high sequence divergence from natural DNA, in vivo measurements show that 57% of the highly-expressed synthetic sequences surpass the expression levels of highly-expressed natural controls. This demonstrates the applicability and relevance of deep generative design to expand our knowledge and control of gene expression regulation in any desired organism, condition or tissue.
Published in DiRROS: 17.07.2024; Views: 442; Downloads: 194
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1319. Diversity of molluscan assemblage in relation to biotic and abiotic variables in brown algal forestsMartina Orlando-Bonaca, Domen Trkov, Katja Klun, Valentina Pitacco, 2022, original scientific article Abstract: Canopy-forming macroalgae, mainly those belonging to the order Fucales, form the so-called brown algal forests, which are among the most productive assemblages in shallow coastal zones. Their vertical, branching canopies increase nearshore primary production, provide nursery areas for juvenile fish, and sustain understory assemblages of smaller algae and both sessile and vagile fauna. The majority of benthic invertebrates inhabiting these forests have larval stages that spend some time floating freely or swimming in the plankton. Therefore, canopy-forming macroalgae play an important role as species collectors related to larval supply and hydrodynamic processes. During the past several decades, brown algal forests have significantly reduced their extension and coverage in the Mediterranean basin, due to multiple interacting natural and anthropogenic pressures, with negative consequences also for the related fauna. The aim of this research was to examine how differences in macrophyte abundance and structure, as well as environmental variables, affect the associated molluscan communities in the shallow northern Adriatic Sea. Sampling sites with well-developed vegetation cover dominated by different canopy-forming species were selected in the shallow infralittoral belt of the northern Adriatic Sea in the spring–summer period of the years 2019 and 2020. Our results confirm the importance of algal forests for molluscan assemblage, with a total of 68 taxa of molluscs found associated with macrophytes. Gastropods showed the highest richness and abundance, followed by bivalves. Mollusc richness and diversity (in terms of biotic indices) were not related with the degree of development of canopy-forming species (in terms of total cover and total volume), nor with the ecological status of benthic macroalgae at different depths. On the contrary, the variability in molluscan taxa abundances was explained by some environmental variables, such as temperature, pH, light, and nitrates concentration.
Keywords: canopy-forming algae, coverage, molluscan assemblage, diversity, abiotic factors, Adriatic Sea
Published in DiRROS: 17.07.2024; Views: 244; Downloads: 210
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