Base editing of general transcription factor Spt15 to enhance yeast stress tolerance
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE160256 | GEO | 2021/07/14
REPOSITORIES: GEO
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Project description:A screening process was used to determine hyperosmotic as well as cold stress tolerance of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at sub lethal levels of NaCl and 4°C. Analysis of gene expression in cells adapted to cold and hyperosmotic stress revealed significant strain specificity in terms of individual gene expression profiles though general patterns of expression within functional gene subsets were largely similar. . Strain ATCC19115 revealed 150 significantly up- and 146 down-regulated genes in cells adapted to both hyperosmotic and cold stress, 82 up- and 56 down-regulated genes were matching in response to both stresses in strain 70-1700, whereas ScottA, a relatively stress-tolerant strain, showed up-regulation of 77 genes whilst 106 genes were significantly down-regulated. Among these homologously expressed genes only 22 were found to be significantly up-regulated and only 7 were down-regulated in all three strains adapted to hyperosmotic stress and cold temperature, suggesting a dominating strain specific response. Overall adaptation to hyperosmotic and low temperature growth conditions in three L. monocytogenes strains were found to have many parallels, especially when examined using a statistically-based ontological approach. Evidence was presented for strong activation of genes associated with protein synthesis, in particular those coding proteins of the ribosome and involved directly in transcription. Genes associated with DNA maintenance; modification to cell envelope, and cell division were also up-regulated. On the other hand strong suppression of genes associated with carbohydrate metabolism and transport as well as flagella assembly was evident in stress adapted cells most likely due to energy preservation via CodY regulon. This suggests that adaptation to these adverse conditions engages similar mechanisms to cope with the induced stressful environments. The microarray component of the experiments had the aim of determining: i) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic salt resistances following adaptation to hyperosmotic stress. ii) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic cold tolerances following adaptation to cold stress. iii) To evaluate common pathways of adaptation to hyperosmotic and cold stresses
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Project description:Here, we investigate the genetic mechanisms that underlie thermal specialization of closely-related vibrios isolated from coastal water at the Beaufort Inlet (Beaufort, NC, USA). This location experiences large seasonal temperature fluctuations (annual range of ~20°C), and a clear seasonal shift in vibrio diversity has been observed (Yung et al. 2015). This previous study suggested that the mechanisms of thermal adaptation apparently differ based on evolutionary timescale: shifts in the temperature of maximal growth occur between deeply branching clades but the shape of the thermal performance curve changes on shorter time scales (Yung et al. 2015). The observed thermal specialization in vibrio populations over relatively short evolutionary time scales indicates that few genes or cellular processes may contribute to the differences in thermal performance between populations. In order to understand the molecular mechanisms that underlie adaptation to local thermal regimes in environmental vibrio populations, we employ genomic and transcriptomic approaches to examine transcriptomic changes that occur within strains grown at their thermal optima and under heat and cold stress. Moreover, we compare two closely-related strains with different laboratory thermal preferences to identify in situ evolutionary responses to different thermal environments in genome content and alleles as well as gene expression.
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Project description:A screening process was used to determine hyperosmotic as well as cold stress tolerance of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at sub lethal levels of NaCl and 4M-BM-0C. Analysis of gene expression in cells adapted to cold and hyperosmotic stress revealed significant strain specificity in terms of individual gene expression profiles though general patterns of expression within functional gene subsets were largely similar. . Strain ATCC19115 revealed 150 significantly up- and 146 down-regulated genes in cells adapted to both hyperosmotic and cold stress, 82 up- and 56 down-regulated genes were matching in response to both stresses in strain 70-1700, whereas ScottA, a relatively stress-tolerant strain, showed up-regulation of 77 genes whilst 106 genes were significantly down-regulated. Among these homologously expressed genes only 22 were found to be significantly up-regulated and only 7 were down-regulated in all three strains adapted to hyperosmotic stress and cold temperature, suggesting a dominating strain specific response. Overall adaptation to hyperosmotic and low temperature growth conditions in three L. monocytogenes strains were found to have many parallels, especially when examined using a statistically-based ontological approach. Evidence was presented for strong activation of genes associated with protein synthesis, in particular those coding proteins of the ribosome and involved directly in transcription. Genes associated with DNA maintenance; modification to cell envelope, and cell division were also up-regulated. On the other hand strong suppression of genes associated with carbohydrate metabolism and transport as well as flagella assembly was evident in stress adapted cells most likely due to energy preservation via CodY regulon. This suggests that adaptation to these adverse conditions engages similar mechanisms to cope with the induced stressful environments. The microarray component of the experiments had the aim of determining: i) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic salt resistances following adaptation to hyperosmotic stress. ii) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic cold tolerances following adaptation to cold stress. iii) To evaluate common pathways of adaptation to hyperosmotic and cold stresses Control cultures (four biological replicates; exception strain ATCC19115 for the 10%NaCl stress which had three biological replicates) were labeled with Cy3 (exception strain ATCC19115 for the 10%NaCl-3 dyeswapped, where the control was labeled with Cy5 and sample with Cy3) for each treatment sample for three different L. monocytogenes strains. Sample cultures (four biological replicates; exception strain ATCC19115 for the 10%NaCl which had three biological replicates) were labeled with Cy5 (exception strain ATCC19115 for the 10%NaCl-3 dyeswapped, where the control was labeled with Cy5 and sample with Cy3) for each treatment sample for three different L. monocytogenes strains. Condition one M-bM-^@M-^Ssub-lethal hyperosmotic stress: Control culture (Cy3-labelled RNA, except ATCC19115 for the 10%NaCl-3 dyeswapped , where the control was labeled with Cy5 and sample with Cy3) - strains were grown to mid exponential growth phase at 25M-BM-0C (in a shaking water bath) in brain-heart infusion broth. Test cultures (Cy5-labelled RNA, except ATCC19115 for the 10%NaCl-3 dyeswapped , where the control was labeled with Cy5 and sample with Cy3) M-bM-^@M-^S strains grown to mid exponential growth at 25M-BM-0C phase (in a shaking water bath) in BHI broth supplemented with 8% additional NaCl (strain 701700), 10% additional NaCl (strain ATCC19115) or 12% additional NaCl (ScottA). Condition two M-bM-^@M-^S cold stress: Control culture (Cy3-labelled RNA) - strains were grown to mid exponential growth phase at 25M-BM-0C (in a shaking water bath) in brain-heart infusion broth. Test cultures (Cy5-labelled RNA) M-bM-^@M-^S strains grown to exponential growth phase at 4M-BM-0C in BHI broth.
2013-05-04 | E-GEOD-46612 | biostudies-arrayexpress
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Project description:Engineering of adenine base editor with improved editing activity across NNN PAMs
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Project description:Recent optimization of CRISPR/Cas9-mediated genome engineering has resulted in the development of base editors that can efficiently mediate C>T and A>G transitions. Combining these genome engineering tools with human adult stem cell (ASC)-derived organoid technology holds promise for disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived hepatocyte, endometrial and intestinal organoids. First, using conventional and evolved Cas9-variants, we show efficacy of both cytosine and adenine base editors and use them to model four hot-spot point mutations in CTNNB1 in hepatocyte organoids. Next, we apply C>T base editors in endometrial organoids to insert nonsense mutations in PTEN and demonstrate tumorigenicity even in the heterozygous state. Furthermore, we use cytosine base editors for simultaneous oncogene activation (PIK3CA) and tumor-suppressor inactivation (APC and TP53). To increase the flexibility of base editor multiplexing, we then combine SpCas9 and SaCas9 base editors for simultaneous C>T and A>G editing at individual target sites. Finally, we show the power of base editor multiplexing by modeling colorectal tumorigenesis in a single step by simultaneously transfecting sgRNA’s targeting four cancer genes.
Seven clonal organoid lines and one bulk wild-type control sample were paired-end whole-genome sequenced using the Illumina Novaseq 6000 system. We sequenced four clonal intestinal organoid lines harbouring engineered TP53 and FBXW7 mutations as well as three lines targeted for oncogenic APC/TP53/PIK3CA/SMAD4 mutations. This WGS showed, as previously reported, a genome-wide increase in C>T mutations due to C>T base editor off-target activity, which is not enriched in predicted off-target regions based on the sgRNA sequences. Furthermore, we confirmed the absence of editing-induced driver mutations and lack of off-target mutational hotspots created by the genomic engineering.
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Project description:CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.
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