scholarly journals An inducible CRISPR-interference library for genetic interrogation of Saccharomyces cerevisiae biology

2020 ◽  
Author(s):  
Amir Momen-Roknabadi ◽  
Panos Oikonomou ◽  
Maxwell Zegans ◽  
Saeed Tavazoie
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Function ◽  
Transcriptional Repression ◽  
Experimental Conditions ◽  
Crispr Interference ◽  
Cellular Processes ◽  
False Discovery ◽  
Genome Wide ◽  
A Genome ◽  
Genome Scale

AbstractGenome-scale CRISPR interference (CRISPRi) is widely utilized to study cellular processes in a variety of organisms. Despite its dominance as a model eukaryote, a genome-wide CRISPRi library, optimized for targeting the Saccharomyces cerevisiae genome, has not been presented to date. We have generated a comprehensive, inducible CRISPRi library, based on spacer design rules optimized for yeast. We have validated this library for genome-wide interrogation of gene function across a variety of applications, including accurate discovery of haploinsufficient genes and identification of enzymatic and regulatory genes involved in adenine and arginine biosynthesis. The comprehensive nature of the library also revealed parameters for optimal transcriptional repression, including upstream distance, nucleosomal occupancy, and strand bias. CRISPRi screens, using this library can identify genes and pathways with high precision and low false discovery rate across a variety of experimental conditions, enabling rapid and reliable genome-wide assessment of gene function and genetic interactions in S.cerevisiae.

scholarly journals An inducible CRISPR interference library for genetic interrogation of Saccharomyces cerevisiae biology

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Amir Momen-Roknabadi ◽  
Panos Oikonomou ◽  
Maxwell Zegans ◽  
Saeed Tavazoie
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Repression ◽  
Nucleosome Occupancy ◽  
Design Parameters ◽  
Experimental Conditions ◽  
Crispr Interference ◽  
Cellular Processes ◽  
Genome Wide ◽  
Reliable Assessment ◽  
A Genome

AbstractGenome-scale CRISPR interference (CRISPRi) is widely utilized to study cellular processes in a variety of organisms. Despite the dominance of Saccharomyces cerevisiae as a model eukaryote, an inducible genome-wide CRISPRi library in yeast has not yet been presented. Here, we present a genome-wide, inducible CRISPRi library, based on spacer design rules optimized for S. cerevisiae. We have validated this library for genome-wide interrogation of gene function across a variety of applications, including accurate discovery of haploinsufficient genes and identification of enzymatic and regulatory genes involved in adenine and arginine biosynthesis. The comprehensive nature of the library also revealed refined spacer design parameters for transcriptional repression, including location, nucleosome occupancy and nucleotide features. CRISPRi screens using this library can identify genes and pathways with high precision and a low false discovery rate across a variety of experimental conditions, enabling rapid and reliable assessment of genetic function and interactions in S. cerevisiae.

scholarly journals Functional genomics in yeast

2007 ◽  
Vol 28 (2) ◽  
pp. 51
Author(s):  
Ian W Dawes ◽  
Geoffrey D Kornfeld ◽  
Gabriel G Perrone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Genomics ◽  
Genome Sequencing ◽  
Genome Sequencing Project ◽  
Sequencing Project ◽  
Cellular Processes ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
And Function

Completion of the Saccharomyces cerevisiae genome sequencing project in 1996 led to an incredible explosion of research on basic cellular processes and has provided the opportunity to determine how genes and their products are regulated and function on a genome-wide scale. The technologies that were developed from this provided an incredible array of tools to study cellular processes in great detail and were a paradigm for developments from subsequent sequencing projects.

scholarly journals A Genome-Wide Screen in Saccharomyces cerevisiae Reveals a Critical Role for Oxidative Phosphorylation in Cellular Tolerance to Lithium Hexafluorophosphate

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 888
Author(s):  
Xuejiao Jin ◽  
Jie Zhang ◽  
Tingting An ◽  
Huihui Zhao ◽  
Wenhao Fu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oxidative Phosphorylation ◽  
Cellular Response ◽  
Critical Role ◽  
Lithium Ion ◽  
Deletion Mutants ◽  
High Concentration ◽  
Genome Wide ◽  
A Genome ◽  
Lithium Hexafluorophosphate

Lithium hexafluorophosphate (LiPF6) is one of the leading electrolytes in lithium-ion batteries, and its usage has increased tremendously in the past few years. Little is known, however, about its potential environmental and biological impacts. In order to improve our understanding of the cytotoxicity of LiPF6 and the specific cellular response mechanisms to it, we performed a genome-wide screen using a yeast (Saccharomyces cerevisiae) deletion mutant collection and identified 75 gene deletion mutants that showed LiPF6 sensitivity. Among these, genes associated with mitochondria showed the most enrichment. We also found that LiPF6 is more toxic to yeast than lithium chloride (LiCl) or sodium hexafluorophosphate (NaPF6). Physiological analysis showed that a high concentration of LiPF6 caused mitochondrial damage, reactive oxygen species (ROS) accumulation, and ATP content changes. Compared with the results of previous genome-wide screening for LiCl-sensitive mutants, we found that oxidative phosphorylation-related mutants were specifically hypersensitive to LiPF6. In these deletion mutants, LiPF6 treatment resulted in higher ROS production and reduced ATP levels, suggesting that oxidative phosphorylation-related genes were important for counteracting LiPF6-induced toxicity. Taken together, our results identified genes specifically involved in LiPF6-modulated toxicity, and demonstrated that oxidative stress and ATP imbalance maybe the driving factors in governing LiPF6-induced toxicity.

scholarly journals A genome-scale metabolic model of Saccharomyces cerevisiae that integrates expression constraints and reaction thermodynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Omid Oftadeh ◽  
Pierre Salvy ◽  
Maria Masid ◽  
Maxime Curvat ◽  
Ljubisa Miskovic ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Expression System ◽  
Biomass Composition ◽  
Industrial Biotechnology ◽  
Overflow Metabolism ◽  
Cellular Expression ◽  
A Genome ◽  
Wide Range ◽  
Eukaryotic Organisms ◽  
Genome Scale

AbstractEukaryotic organisms play an important role in industrial biotechnology, from the production of fuels and commodity chemicals to therapeutic proteins. To optimize these industrial systems, a mathematical approach can be used to integrate the description of multiple biological networks into a single model for cell analysis and engineering. One of the most accurate models of biological systems include Expression and Thermodynamics FLux (ETFL), which efficiently integrates RNA and protein synthesis with traditional genome-scale metabolic models. However, ETFL is so far only applicable for E. coli. To adapt this model for Saccharomyces cerevisiae, we developed yETFL, in which we augmented the original formulation with additional considerations for biomass composition, the compartmentalized cellular expression system, and the energetic costs of biological processes. We demonstrated the ability of yETFL to predict maximum growth rate, essential genes, and the phenotype of overflow metabolism. We envision that the presented formulation can be extended to a wide range of eukaryotic organisms to the benefit of academic and industrial research.

scholarly journals Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

2014 ◽  
Vol 42 (15) ◽  
pp. 9838-9853 ◽  
Author(s):  
Saeed Kaboli ◽  
Takuya Yamakawa ◽  
Keisuke Sunada ◽  
Tao Takagaki ◽  
Yu Sasano ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Interaction Network ◽  
Essential Genes ◽  
Genetic Interactions ◽  
Lethal Gene ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale

Abstract Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation

2021 ◽  
Vol 14 (694) ◽  
pp. eabe0387
Author(s):  
Orna Ernst ◽  
Jing Sun ◽  
Bin Lin ◽  
Balaji Banoth ◽  
Michael G. Dorrington ◽  
...  
Keyword(s):  
Nucleoside Diphosphate Kinase ◽  
Multiple Roles ◽  
Metabolic Reprogramming ◽  
Inflammasome Activation ◽  
Ros Production ◽  
Genome Wide ◽  
A Genome ◽  
Mouse Macrophages ◽  
Genome Scale ◽  
Mitochondrial Dna Synthesis

Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor–associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D–dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.

scholarly journals Chromatin architectural proteins regulate flowering time by precluding gene looping

2021 ◽  
Vol 7 (24) ◽  
pp. eabg3097
Author(s):  
Bo Zhao ◽  
Yanpeng Xi ◽  
Junghyun Kim ◽  
Sibum Sung
Keyword(s):  
Chromatin Structure ◽  
Cellular Processes ◽  
Genome Wide ◽  
A Genome ◽  
Evolutionarily Conserved ◽  
Architectural Proteins ◽  
Floral Repressor ◽  
Flanking Regions ◽  
Genome Wide Study

Chromatin structure is critical for gene expression and many other cellular processes. In Arabidopsis thaliana, the floral repressor FLC adopts a self-loop chromatin structure via bridging of its flanking regions. This local gene loop is necessary for active FLC expression. However, the molecular mechanism underlying the formation of this class of gene loops is unknown. Here, we report the characterization of a group of linker histone-like proteins, named the GH1-HMGA family in Arabidopsis, which act as chromatin architecture modulators. We demonstrate that these family members redundantly promote the floral transition through the repression of FLC. A genome-wide study revealed that this family preferentially binds to the 5′ and 3′ ends of gene bodies. The loss of this binding increases FLC expression by stabilizing the FLC 5′ to 3′ gene looping. Our study provides mechanistic insights into how a family of evolutionarily conserved proteins regulates the formation of local gene loops.

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