Construction and evaluation of gRNA arrays for multiplex CRISPR-Cas9.

Endonuclease system CRISPR-Cas9 represents a powerful toolbox for the budding yeast's Saccharomyces cerevisiae genome perturbation. The resulting double-strand breaks are preferentially repaired via highly efficient homologous recombination, which subsequently leads to marker-free genome editing. The goal of this study was to evaluate precise targeting of multiple loci simultaneously. To construct an array of independently expressing guide RNAs (gRNAs), the genes encoding them were assembled through a BioBrick construction procedure. We designed a multiplex CRISPR-Cas9 system for targeting 6 marker genes, whereby the gRNA array was expressed from a single plasmid. To evaluate the performance of the gRNA array, the activity of the designed system was assessed by the success rate of the introduction of perturbations within the target loci: successful gRNA expression, followed by target DNA double-strand breaks formation and their repair by homologous recombination led to premature termination of the coding sequence of the marker genes, resulting in the prevention of growth of the transformants on the corresponding selection media. In conclusion, we successfully introduced up to five simultaneous perturbations within single cells of yeast S. cerevisiae using the multiplex CRISPR-Cas9 system. While this has been done before, we here present an alternative sequential BioBrick assembly with the capability to accommodate many highly similar gRNA-expression cassettes, and an exhaustive evaluation of their performance.

Unilateral ends-out gene targeting increases mistargeting through supporting extensive single-strand assimilation.

Ends-out gene targeting enables the swapping of endogenous alleles with exogenous ones through homologous recombination which bears great implications both fundamental and applicable. To address the recombination mechanism(s) behind it, an experimental system was designed to distinguish between a possible (but rarely active) unilateral and the expected bilateral targeting in the yeast Saccharomyces cerevisiae in which the proportions of the two alternative genetic outcomes are conceived to mirror the probabilities of the two scenarios. The quantitative analysis showed that the bilateral targeting was expectedly predominant. However, an analogous comparative analysis on a different experimental set suggested a prevalence of unilateral targeting unveiling an uncertainty whether the extensively resected targeting modules only mimic unilateral invasion. Based on this, a comprehensive qualitative analysis was conducted revealing a single basic ends-out gene targeting mechanism composed of two intertwined pathways differing in the way how the homologous invasion is initiated and/or the production of the intermediates is conducted. This study suggests that bilateral targeting lowers mistargeting plausibly by limiting strand assimilation, unlike unilateral targeting which may initiate extensive strand assimilation producing intermediates capable of supporting multiple genetic outcomes which leads to mistargeting. Some of these outcomes can also be produced by mimicking unilateral invasion.

Biolistic transformation of the yeast Saccharomyces cerevisiae mitochondrial DNA.

The insertion of genes into mitochondria by biolistic transformation is currently only possible in the yeast Saccharomyces cerevisiae and the algae Chlamydomonas reinhardtii. The fact that S. cerevisiae mitochondria can exist with partial (ρ- mutants) or complete deletions (ρ0 mutants) of mitochondrial DNA (mtDNA), without requiring a specific origin of replication, enables the propagation of exogenous sequences. Additionally, mtDNA in this organism undergoes efficient homologous recombination, making it well-suited for genetic manipulation. In this review, we present a summarized historical overview of the development of biolistic transformation and discuss iconic applications of the technique. We also provide a detailed example on how to obtain transformants with recombined foreign DNA in their mitochondrial genome.

Polymorphism of Saccharomyces cerevisiae Strains in DNA Metabolism Genes.

Baker's yeast, S. cerevisiae, is an excellent model organism exploited for molecular genetic studies of the mechanisms of genome stability in eukaryotes. Genetic peculiarities of commonly used yeast strains impact the processes of DNA replication, repair, and recombination (RRR). We compared the genomic DNA sequence variation of the five strains that are intensively used for RRR studies. We used yeast next-generation sequencing data to detect the extent and significance of variation in 183 RRR genes. We present a detailed analysis of the differences that were found even in closely related strains. Polymorphisms of common yeast strains should be considered when interpreting the outcomes of genome stability studies, especially in cases of discrepancies between laboratories describing the same phenomena.

Insertion orientation within the cassette affects gene-targeting success during ends-out recombination in the yeast Saccharomyces cerevisiae.

Gene-targeting is one of the most important molecular tools for genomic manipulations for research and industrial purposes. However, many factors influence targeting fidelity undermining the efforts for accurate, fast, and reliable construction of genetically modified yeast strains. Therefore, it is of great academic interest that we uncover as many as possible parameters affecting the recombination mechanisms that enable targeting. Since usually, researchers choose the orientation of the insertion (marker) within the module at random, it seemed interesting to see whether the same module will achieve essentially the same targeting efficiency when the same marker was oriented alternatively concerning the same target gene. Thus, two loci (URA3 and LEU2) and one allele (ura3-52) in a haploid yeast genetic background were targeted by artificial modules bearing homologous insertions in alternative orientations being flanked by long asymmetrical targeting homology to either replace or disrupt a genomic target. Results showed that insertion orientation within the targeting module strongly influences targeting in yeast, regardless of the targeting approach.

RAD52 influences the effect of BRCA1/2 missense variants on homologous recombination and gene reversion in Saccharomyces cerevisiae.

The breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, are key players in the homologous recombination (HR) repair pathway and act as tumor suppressors by maintaining genome stability. The yeast Saccharomyces cerevisiae has no BRCA1/2 homolog; however, a number of HR genes are evolutionary conserved between human and yeast. Among them, RAD52 is involved in DNA double strand break (DSB) repair by HR, and promotes genome stability. We previously reported that the heterologous expression of cancer-associated BRCA1/2 missense variants in growing yeast cultures affects both spontaneous HR and gene reversion (GR) suggesting that yeast could be a reliable system to assess the functional impact of variants. Because inhibition of Rad52p is lethal in BRCA1/2 mutated tumors, and Rad52p is conserved between humans and yeast, we asked if the effect of BRCA1/2 variants on HR and GR could be affected by loss of RAD52. We found that the rad52∆ mutation predominantly suppressed the stimulation of HR in yeast by pathogenic BRCA1 variants but also facilitated increased GR by pathogenic variants. Conversely, the rad52∆ mutation stimulated HR by a pathogenic BRCA2 variant in yeast but had no effect on GR. These results demonstrate a functional interplay between the pathogenic BRCA1/2 variants and Rad52p in budding yeast, supporting the use of budding yeast as a suitable system for evaluating potential chemotherapeutic strategies.

Validation of the Pathogenic Effect of IGHMBP2 Gene Mutations Based on Yeast S. cerevisiae Model.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a heritable neurodegenerative disease characterized by rapid respiratory failure within the first months of life and progressive muscle weakness and wasting. Although the causative gene, IGHMBP2, is well defined, information on IGHMBP2 mutations is not always sufficient to diagnose particular patients, as the gene is highly polymorphic and the pathogenicity of many gene variants is unknown. In this study, we generated a simple yeast model to establish the significance of IGHMBP2 variants for disease development, especially those that are missense mutations. We have shown that cDNA of the human gene encodes protein which is functional in yeast cells and different pathogenic mutations affect this functionality. Furthermore, there is a correlation between the phenotype estimated in in vitro studies and our results, indicating that our model may be used to quickly and simply distinguish between pathogenic and non-pathogenic mutations identified in IGHMBP2 in patients.

Novel Approach in the Construction of Bioethanol-Producing Saccharomyces cerevisiae Hybrids§.

Bioethanol production from lignocellulosic hydrolysates requires a producer strain that tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous intraspecies hybrid diploids of Saccharomyces cerevisiae by crossing two natural S. cerevisiae isolates, YIIc17_E5 and UWOPS87-2421, a good ethanol producer found in wine and a strain from the flower of the cactus Opuntia megacantha resistant to inhibitors found in lignocellulosic hydrolysates, respectively. Hybrids grew faster than parental strains in the absence and in the presence of acetic and levulinic acids and 2-furaldehyde, inhibitors frequently found in lignocellulosic hydrolysates, and the overexpression of YAP1 gene increased their survival. Furthermore, although originating from the same parental strains, hybrids displayed different fermentative potential in a CO2 production test, suggesting genetic variability that could be used for further selection of desirable traits. Therefore, our results suggest that the construction of intraspecies hybrids coupled with the use of genetic engineering techniques is a promising approach for improvement or development of new biotechnologically relevant strains of S. cerevisiae. Moreover, it was found that the success of gene targeting (gene targeting fidelity) in natural S. cerevisiae isolates (YIIc17_E5α and UWOPS87-2421α) was strikingly lower than in laboratory strains and the most frequent off-targeting event was targeted chromosome duplication.

Tum1 is involved in the metabolism of sterol esters in Saccharomyces cerevisiae.

BACKGROUND: The only hitherto known biological role of yeast Saccharomyces cerevisiae Tum1 protein is in the tRNA thiolation pathway. The mammalian homologue of the yeast TUM1 gene, the thiosulfate sulfurtransferase (a.k.a. rhodanese) Tst, has been proposed as an obesity-resistance and antidiabetic gene. To assess the role of Tum1 in cell metabolism and the putative functional connection between lipid metabolism and tRNA modification, we analysed evolutionary conservation of the rhodanese protein superfamily, investigated the role of Tum1 in lipid metabolism, and examined the phenotype of yeast strains expressing the mouse homologue of Tum1, TST. RESULTS: We analysed evolutionary relationships in the rhodanese superfamily and established that its members are widespread in bacteria, archaea and in all major eukaryotic groups. We found that the amount of sterol esters was significantly higher in the deletion strain tum1Δ than in the wild-type strain. Expression of the mouse TST protein in the deletion strain did not rescue this phenotype. Moreover, although Tum1 deficiency in the thiolation pathway was complemented by re-introducing TUM1, it was not complemented by the introduction of the mouse homologue Tst. We further showed that the tRNA thiolation pathway is not involved in the regulation of sterol ester content in S. cerevisiae, as overexpression of the tEUUC, tKUUU and tQUUG tRNAs did not rescue the lipid phenotype in the tum1Δ deletion strain, and, additionally, deletion of the key gene for the tRNA thiolation pathway, UBA4, did not affect sterol ester content. CONCLUSIONS: The rhodanese superfamily of proteins is widespread in all organisms, and yeast TUM1 is a bona fide orthologue of mammalian Tst thiosulfate sulfurtransferase gene. However, the mouse TST protein cannot functionally replace yeast Tum1 protein, neither in its lipid metabolism-related function, nor in the tRNA thiolation pathway. We show here that Tum1 protein is involved in lipid metabolism by decreasing the sterol ester content in yeast cells, and that this function of Tum1 is not exerted through the tRNA thiolation pathway, but through another, currently unknown pathway.

Using yeast to model calcium-related diseases: example of the Hailey-Hailey disease.

Cross-complementation studies offer the possibility to overcome limitations imposed by the inherent complexity of multicellular organisms in the study of human diseases, by taking advantage of simpler model organisms like the budding yeast Saccharomyces cerevisiae. This review deals with, (1) the use of S. cerevisiae as a model organism to study human diseases, (2) yeast-based screening systems for the detection of disease modifiers, (3) Hailey-Hailey as an example of a calcium-related disease, and (4) the presentation of a yeast-based model to search for chemical modifiers of Hailey-Hailey disease. The preliminary experimental data presented and discussed here show that it is possible to use yeast as a model system for Hailey-Hailey disease and suggest that in all likelihood, yeast has the potential to reveal candidate drugs for the treatment of this disorder. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Respiratory complex III dysfunction in humans and the use of yeast as a model organism to study mitochondrial myopathy and associated diseases.

The bc1 complex or complex III is a central component of the aerobic respiratory chain in prokaryotic and eukaryotic organisms. It catalyzes the oxidation of quinols and the reduction of cytochrome c, establishing a proton motive force used to synthesize adenosine triphosphate (ATP) by the F1Fo ATP synthase. In eukaryotes, the complex III is located in the inner mitochondrial membrane. The genes coding for the complex III have a dual origin. While cytochrome b is encoded by the mitochondrial genome, all the other subunits are encoded by the nuclear genome. In this review, we compile an exhaustive list of the known human mutations and associated pathologies found in the mitochondrially-encoded cytochrome b gene as well as the fewer mutations in the nuclear genes coding for the complex III structural subunits and accessory proteins such as BCS1L involved in the assembly of the complex III. Due to the inherent difficulties of studying human biopsy material associated with complex III dysfunction, we also review the work that has been conducted to study the pathologies with the easy to handle eukaryotic microorganism, the yeast Saccharomyces cerevisiae. Phenotypes, biochemical data and possible effects due to the mutations are also discussed in the context of the known three-dimensional structure of the eukaryotic complex III. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Functional interplay between Mediator and RSC chromatin remodeling complex controls nucleosome-depleted region maintenance at promoters.

Chromatin organization is crucial for transcriptional regulation in eukaryotes. Mediator is an essential and conserved co-activator thought to act in concert with chromatin regulators. However, it remains largely unknown how their functions are coordinated. Here, we provide evidence in the yeast Saccharomyces cerevisiae that Mediator establishes physical contact with RSC (Remodels the Structure of Chromatin), a conserved and essential chromatin remodeling complex that is crucial for nucleosome-depleted region (NDR) formation. We determine the role of Mediator-RSC interaction in their chromatin binding, nucleosome occupancy, and transcription on a genomic scale. Mediator and RSC co-localize on wide NDRs of promoter regions, and specific Mediator mutations affect nucleosome eviction and TSS-associated +1 nucleosome stability. This work shows that Mediator contributes to RSC remodeling function to shape NDRs and maintain chromatin organization on promoter regions. It will help in our understanding of transcriptional regulation in the chromatin context relevant for severe diseases.

The effect of live yeast Saccharomyces cerevisiae as probiotic supply on growth performance, feed intake, ruminal pH and fermentation in fattening calves.

BACKGROUND: Live yeast Saccharomyces cerevisiae has been used for a long time in ruminant feed as a probiotic supply on the intensifying system to avoid acidosis. OBJECTIVES: This study investigated the effects of addition of live yeast S. cerevisiae in calf feed on growth, rumen pH and in vitro digestibility. METHODS: Sixteen Holstein calves were divided into two homogeneous groups corresponding to body weight. The ration comprises wheat straw 5 kg dry matter (DM)/calf/day and 8 kg DM/calf/day concentrate for the control group C and for the group LY. Each calf of the live yeast group LY gets more than C group, 28 g/calf/day of live yeast S. cerevisiae powder on the concentrate. RESULTS: This supplementation improves significantly (p < 0.003) the mean daily gain during the trial (ADG) with 400 g/calf. A notable increase (p < 0.004) was seen in final body weight gain (FWG) with 39.1 kg/calf. The live yeast supplementation decreases the feed intake and significantly (p < 0.05) the feed conversion rate (FCR) average. The live yeast S. cerevisiae as probiotic supply in ruminant feed improves the growth performance and feed efficiency in fattening calves. CONCLUSIONS: In conclusion, we mentioned that live yeast supply induces a considerable advance in growth performance for calves.

Dataset for suppressors of amyloid-ß toxicity and their functions in recombinant protein production in yeast.

The production of recombinant proteins at high levels often induces stress-related phenotypes by protein misfolding or aggregation. These are similar to those of the yeast Alzheimer's disease (AD) model in which amyloid-ß peptides (Aß42) were accumulated [1], [2]. We have previously identified suppressors of Aß42 cytotoxicity via the genome-wide synthetic genetic array (SGA) [3] and here we use them as metabolic engineering targets to evaluate their potentiality on recombinant protein production in yeast Saccharomyces cerevisiae. In order to investigate the mechanisms linking the genetic modifications to the improved recombinant protein production, we perform systems biology approaches (transcriptomics and proteomics) on the resulting strain and intermediate strains. The RNAseq data are preprocessed by the nf-core/RNAseq pipeline and analyzed using the Platform for Integrative Analysis of Omics (PIANO) package [4]. The quantitative proteome is analyzed on an Orbitrap Fusion Lumos mass spectrometer interfaced with an Easy-nLC1200 liquid chromatography (LC) system. LC-MS data files are processed by Proteome Discoverer version 2.4 with Mascot 2.5.1 as a database search engine. The original data presented in this work can be found in the research paper titled "Suppressors of Amyloid-ß Toxicity Improve Recombinant Protein Production in yeast by Reducing Oxidative Stress and Tuning Cellular Metabolism", by Chen et al. [5].

Purification and characterization of protease M, a yeast mitochondrial nucleotide-stimulated metal protease: its identification as CYM1 gene product, a mitochondrial presequence peptidase.

A chelator-sensitive protease in the mitochondrial matrix of the yeast, Saccharomyces cerevisiae (Biochem. Biophys. Res. Commun. 144, 277, 1987), was purified and characterized. The purified enzyme, termed protease M, specifically hydrolyzes peptide substrates on the N-side of the paired basic residues. When mastoparan was used as substrate, it cleaved Ala8-Leu9 and Lys11-Lys12 bonds as well as the N-side of Lys11-Lys12 residues. Nucleotide triphosphates stimulated the activity 3-fold at 2.5 mM. The genomic DNA sequence showed that protease M was a gene product of CYM1 known as mitochondrial presequence protease homologue in S. cerevisiae, encoding a 989-amino acid-long precursor protein. The N-terminal sequence of the purified enzyme indicated that protease M has 16-residue signal sequence and the 'mature' protein consists of 973 amino acids with a molecular mass of 110 kDa. Protease M contained consensus sequence motifs of ATP-binding site very near the carboxyl terminus. The alignment of the two ATP-binding motifs is an inverted version of the common alignment. Gene disruption of the enzyme generates mixed subunits in tetrameric MnSOD formed with 23-kDa mature and 24-kDa partial presequence-containing subunits. This report describes newly identified enzyme properties of the CYM1 gene product, protease M and abnormal MnSOD complex formation of the disruption mutant.

Global Comparative Label-Free Yeast Proteome Analysis by LC-MS/MS After High-pH Reversed-Phase Peptide Fractionation Using Solid-Phase Extraction Cartridges.

Discovery-driven comparative proteomics employing the bottom-up strategy with label-free quantification on high-resolution mass analyzers like an Orbitrap in a hybrid instrument has the capacity to reveal unique biological processes in the context of plant metabolic engineering. However, proteins are very heterogeneous in nature with a wide range of expression levels, and overall coverage may be suboptimal regarding both the number of protein identifications and sequence coverage of the identified proteins using conventional data-dependent acquisitions without sample fractionation before online nanoflow liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS). In this chapter, we detail a simple and robust method employing high-pH reversed-phase (HRP) peptide fractionation using solid-phase extraction cartridges for label-free proteomic analyses. Albeit HRP fractionation separates peptides according to their hydrophobicity like the subsequent nanoflow gradient reversed-phased LC relying on low pH mobile phase, the two methods are orthogonal. Presented here as a protocol with yeast (Saccharomyces cerevisiae) as a frequently used model organism and hydrogen peroxide to exert cellular stress and survey its impact compared to unstressed control as an example, the described workflow can be adapted to a wide range of proteome samples for applications to plant metabolic engineering research.

Quality-controlled ceramide-based GPI-anchored protein sorting into selective ER exit sites.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) exit the endoplasmic reticulum (ER) through a specialized export pathway in the yeast Saccharomyces cerevisiae. We have recently shown that a very-long acyl chain (C26) ceramide present in the ER membrane drives clustering and sorting of GPI-APs into selective ER exit sites (ERES). Now, we show that this lipid-based ER sorting also involves the C26 ceramide as a lipid moiety of GPI-APs, which is incorporated into the GPI anchor through a lipid-remodeling process after protein attachment in the ER. Moreover, we also show that a GPI-AP with a C26 ceramide moiety is monitored by the GPI-glycan remodelase Ted1, which, in turn, is required for receptor-mediated export of GPI-APs. Therefore, our study reveals a quality-control system that ensures lipid-based sorting of GPI-APs into selective ERESs for differential ER export, highlighting the physiological need for this specific export pathway.

Model Electrochemical Biosensor for the Detection of Methanol in Aqueous Solutions with Yeast Cells.

An electrochemical device that serves as a model biosensor and contains yeast Saccharomyces cerevisiae as the active biological element was developed. Different configurations of the electrochemical cells were assembled and tested. Stainless steel was used in the electrochemical cell composition process and the surface of this metal electrode was modified with a thin layer of WO3 if necessary. The yeast Saccharomyces cerevisiae was adhered to the working electrode. The resulting model biosensor was then used to monitor the response to a 10% CH3OH. For detection of biological activity, the electrochemical impedance spectroscopy (EIS) method was applied with a portable potentiostat/galvanostat, where the Bode and the Nyquist plots were interpreted. The stability of the device was beforehand determined by measuring the open circuit potential (OCP). The topography of the electrodes was inspected using the techniques of scanning electron microscopy and optical microscopy. The investigated model biosensor serves as a case study for the development of more complex biosensors that utilize living cells as the active layer.

Data from crosslinking and analysis of cDNAs (CRAC) of Nab3 in yeast cells expressing a circular ncRNA decoy.

Pervasive transcription originating from the ubiquitous activity of RNA Polymerase II (RNAPII) generates a vast mass of non-coding RNAs (ncRNAs) that represent a potential harm to gene expression. In the compact genome of the yeast Saccharomyces cerevisiae, the main genomewide safeguard against pervasive ncRNAs is the Nrd1-Nab3-Sen1 (NNS) complex, composed of two RNA-binding proteins (Nrd1 and Nab3) and the helicase Sen1. The NNS complex directs transcription termination of ncRNA genes and promotes the rapid degradation of pervasive transcripts from yeast nuclei through its physical and functional coupling to the nuclear RNA exosome. We have recently shown that inhibition of the exosome in yeast cells leads to the accumulation of ncRNAs complexed with Nab3 and Nrd1, decreasing recycling of these termination factors to sites of transcription and inducing global termination defects at NNS targets. Consistent with the notion that ncRNAs out-titrate Nab3 and Nrd1 termination factors, we have shown that a similar genomewide termination impairment could be achieved by expressing a circular RNA decoy containing a Nab3 binding target [1]. In relation to this previous research article, here we expand our observations on the effect of the circular RNA decoy on NNS termination. We aimed at verifying that the Nab3 binding sequence present on the decoy is indeed efficiently sequestering Nab3 as intended by design, leading to the expected decrease of Nab3 binding on NNS targets. We employed the crosslinking and cDNA analysis protocol (CRAC) on yeast cells expressing the circular ncRNA decoy or a control construct. We present data from high-resolution genomewide RNA binding of Nab3 in three independent biological replicates of these S.cerevisiae cells, normalized by spiked-in S.pombe lysates. These data allow the useful assessment of the extent of co-transcriptional binding decrease of Nab3 by decoy ncRNA titration and will be valuable for further analyses of NNS targeting mechanisms.

Size-dependent antirecombinogenic effect of short spacers on palindrome recombinogenicity.

Palindromic sequences in DNA can instigate genetic recombination and genome instability, which can result in devastating conditions such as the Emmanuel syndrome. Palindrome recombinogenicity increases with its size and sequence similarity between palindrome arms, while quasipalindromes with long spacers are less recombinogenic. However, the minimal spacer length, which could reduce or abolish palindrome recombinogenicity in the eukaryotic genome, was never determined. Therefore, we constructed a series of palindromes containing spacers of lengths ranging from 0 (perfect palindrome) to 10 bp and tested their recombinogenicity in yeast Saccharomyces cerevisiae. We found that a 7 bp spacer significantly reduces 126 bp palindrome recombinogenicity, while a 10 bp spacer completely stabilizes palindromes up to 150 bp long. Additionally, we showed that palindrome stimulated recombination rate is not dependent on Mus81 and Yen1 endonucleases. We also compared the recombinogenicity of a perfect 126 bp palindrome and a corresponding quasipalindrome consisting of the same palindrome arms with a stabilising 10 bp spacer in sgs1Δ and rad27Δ backgrounds, since both Sgs1 helicase and Rad27 endonuclease are implicated in preventing hairpin formation at palindromic sequences during replication.