scholarly journals Genome-wide Cas9 binding specificity in Saccharomyces cerevisiae

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9442 ◽  
Author(s):  
Zachary J. Waldrip ◽  
Piroon Jenjaroenpun ◽  
Oktawia DeYoung ◽  
Intawat Nookaew ◽  
Sean D. Taverna ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Specificity ◽  
Genomic Locus ◽  
Site Specific ◽  
Guide Rna ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Chromosome Iii ◽  
Genomic Editing

The CRISPR system has become heavily utilized in biomedical research as a tool for genomic editing as well as for site-specific chromosomal localization of specific proteins. For example, we developed a CRISPR-based methodology for enriching a specific genomic locus of interest for proteomic analysis in Saccharomyces cerevisiae, which utilized a guide RNA-targeted, catalytically dead Cas9 (dCas9) as an affinity reagent. To more comprehensively evaluate the genomic specificity of using dCas9 as a site-specific tool for chromosomal studies, we performed dCas9-mediated locus enrichment followed by next-generation sequencing on a genome-wide scale. As a test locus, we used the ARS305 origin of replication on chromosome III in S. cerevisiae. We found that enrichment of this site is highly specific, with virtually no off-target enrichment of unique genomic sequences. The high specificity of genomic localization and enrichment suggests that dCas9-mediated technologies have promising potential for site-specific chromosomal studies in organisms with relatively small genomes such as yeasts.

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.

scholarly journals Integrated cross-study datasets of genetic dependencies in cancer

2020 ◽  
Author(s):  
Clare Pacini ◽  
Joshua M. Dempster ◽  
Isabella Boyle ◽  
Emanuel Gonçalves ◽  
Hanna Najgebauer ◽  
...  
Keyword(s):  
Statistical Power ◽  
Batch Effects ◽  
Copy Number Alterations ◽  
Cancer Genetic ◽  
Guide Rna ◽  
Large Panels ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
The Individual

AbstractCRISPR-Cas9 viability screens are increasingly performed at a genome-wide scale across large panels of cell lines to identify new therapeutic targets for precision cancer therapy. Integrating the datasets resulting from these studies is necessary to adequately represent the heterogeneity of human cancers and to assemble a comprehensive map of cancer genetic vulnerabilities. Here, we integrated the two largest public independent CRISPR-Cas9 screens performed to date (at the Broad and Sanger institutes) by assessing, comparing, and selecting methods for correcting biases due to heterogeneous single guide RNA efficiency, gene-independent responses to CRISPR-Cas9 targeting originated from copy number alterations, and experimental batch effects. Our integrated datasets recapitulate findings from the individual datasets, provide greater statistical power to cancer- and subtype-specific analyses, unveil additional biomarkers of gene dependency, and improve the detection of common essential genes. We provide the largest integrated resources of CRISPR-Cas9 screens to date and the basis for harmonizing existing and future functional genetics datasets.

scholarly journals Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes

2021 ◽  
Vol 220 (2) ◽  
Author(s):  
Gil Kanfer ◽  
Shireen A. Sarraf ◽  
Yaakov Maman ◽  
Heather Baldwin ◽  
Eunice Dominguez-Martin ◽  
...  
Keyword(s):  
Neural Network ◽  
Fluorescent Protein ◽  
Network Models ◽  
Neural Network Models ◽  
Guide Rna ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Screening Platform ◽  
Cellular Phenotypes

Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9-KRAB (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by using microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated and isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty-one of the 64 hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-photoswitchable screening (AI-PS), offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.

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.

Alternative Isoform Detection Using Exon Arrays

2009 ◽  
pp. 262-277
Author(s):  
Jacek Majewski
Keyword(s):  
Human Populations ◽  
Genomic Locus ◽  
Exon Arrays ◽  
Eukaryotic Genes ◽  
Genome Wide ◽  
A Genome ◽  
Potential Problems ◽  
Recent Developments ◽  
Wide Scale ◽  
Alternative Isoforms

Eukaryotic genes have the ability to produce several distinct products from a single genomic locus. Recent developments in microarray technology allow monitoring of such isoform variation at a genome-wide scale. In our research, we have used Affymetrix Exon Arrays to detect variation in alternative splicing, initiation of transcription, and polyadenylation among humans. We demonstrated that such variation is common in human populations and has an underlying genetic component. Here, we use our study to illustrate the use of Exon Arrays to detect alternative isoforms, to outline the analysis involved, and to point out potential problems that may be encountered by researchers using this technology.

scholarly journals The conserved elongation factor Spn1 is required for normal transcription, histone modifications, and splicing in Saccharomyces cerevisiae

2020 ◽  
Vol 48 (18) ◽  
pp. 10241-10258 ◽  
Author(s):  
Natalia I Reim ◽  
James Chuang ◽  
Dhawal Jain ◽  
Burak H Alver ◽  
Peter J Park ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Elongation Factor ◽  
Mrna Levels ◽  
Chromatin Dynamics ◽  
Genome Wide ◽  
A Genome ◽  
Highly Expressed Genes ◽  
Wide Scale

Abstract Spn1/Iws1 is a conserved protein involved in transcription and chromatin dynamics, yet its general in vivo requirement for these functions is unknown. Using a Spn1 depletion system in Saccharomyces cerevisiae, we demonstrate that Spn1 broadly influences several aspects of gene expression on a genome-wide scale. We show that Spn1 is globally required for normal mRNA levels and for normal splicing of ribosomal protein transcripts. Furthermore, Spn1 maintains the localization of H3K36 and H3K4 methylation across the genome and is required for normal histone levels at highly expressed genes. Finally, we show that the association of Spn1 with the transcription machinery is strongly dependent on its binding partner, Spt6, while the association of Spt6 and Set2 with transcribed regions is partially dependent on Spn1. Taken together, our results show that Spn1 affects multiple aspects of gene expression and provide additional evidence that it functions as a histone chaperone in vivo.

scholarly journals Integrated cross-study datasets of genetic dependencies in cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Clare Pacini ◽  
Joshua M. Dempster ◽  
Isabella Boyle ◽  
Emanuel Gonçalves ◽  
Hanna Najgebauer ◽  
...  
Keyword(s):  
Statistical Power ◽  
Batch Effects ◽  
Copy Number Alterations ◽  
Cancer Genetic ◽  
Guide Rna ◽  
Large Panels ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
The Individual

AbstractCRISPR-Cas9 viability screens are increasingly performed at a genome-wide scale across large panels of cell lines to identify new therapeutic targets for precision cancer therapy. Integrating the datasets resulting from these studies is necessary to adequately represent the heterogeneity of human cancers and to assemble a comprehensive map of cancer genetic vulnerabilities. Here, we integrated the two largest public independent CRISPR-Cas9 screens performed to date (at the Broad and Sanger institutes) by assessing, comparing, and selecting methods for correcting biases due to heterogeneous single-guide RNA efficiency, gene-independent responses to CRISPR-Cas9 targeting originated from copy number alterations, and experimental batch effects. Our integrated datasets recapitulate findings from the individual datasets, provide greater statistical power to cancer- and subtype-specific analyses, unveil additional biomarkers of gene dependency, and improve the detection of common essential genes. We provide the largest integrated resources of CRISPR-Cas9 screens to date and the basis for harmonizing existing and future functional genetics datasets.

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.

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