scholarly journals RELATe enables genome-scale engineering in fungal genomics

2020 ◽  
Vol 6 (38) ◽  
pp. eabb8783
Author(s):  
Zhongming Li ◽  
Kwang Sik Kim
Keyword(s):  
High Throughput Screening ◽  
Traditional Approach ◽  
Central Nervous System Infection ◽  
Cost Effective ◽  
Fungal Species ◽  
Model Organisms ◽  
Simple Method ◽  
Guide Rna ◽  
Fungal Genomics ◽  
Genome Scale

CRISPR-Cas9–based screening with single-guide RNA (sgRNA) libraries has emerged as a revolutionary tool for comprehensive analysis of genetic elements. However, genome-scale sgRNA libraries are currently available only in a few model organisms. The traditional approach is to synthesize thousands to tens of thousands of sgRNAs, which is laborious and expensive. We have developed a simple method, RELATe (restriction/ligation coupled with Agrobacterium-mediated transformation), to generate sgRNA libraries from 10 μg of genomic DNA, targeting over 98% of the protein-coding genes in the human fungal pathogen Cryptococcus neoformans. Functional screens identified 142 potential C. neoformans genes contributing to blood-brain barrier penetration. We selected two cryptococcal genes, SFP1 and WDR1, for a proof-of-concept demonstration that RELATe-identified genes are relevant to C. neoformans central nervous system infection. Our RELATe method can be used in many other fungal species and is powerful and cost-effective for genome-wide high-throughput screening for elucidating functional genomics.

scholarly journals On-target activity predictions enable improved CRISPR–dCas9 screens in bacteria

2020 ◽  
Vol 48 (11) ◽  
pp. e64-e64 ◽  
Author(s):  
Alicia Calvo-Villamañán ◽  
Jérome Wong Ng ◽  
Rémi Planel ◽  
Hervé Ménager ◽  
Arthur Chen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cost Effective ◽  
Target Sequence ◽  
Guide Rna ◽  
E Coli ◽  
Genome Wide ◽  
High Throughput Screens ◽  
Target Activity ◽  
Target Effects

Abstract The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology to probe gene function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack a good understanding of the design rules that determine on-target activity for dCas9. Taking advantage of high-throughput screening data, we fit a model to predict the ability of dCas9 to block the RNA polymerase based on the target sequence, and validate its performance on independently generated datasets. We further design a novel genome wide guide RNA library for E. coli MG1655, EcoWG1, using our model to choose guides with high activity while avoiding guides which might be toxic or have off-target effects. A screen performed using the EcoWG1 library during growth in rich medium improved upon previously published screens, demonstrating that very good performances can be attained using only a small number of well designed guides. Being able to design effective, smaller libraries will help make CRISPRi screens even easier to perform and more cost-effective. Our model and materials are available to the community through crispr.pasteur.fr and Addgene.

Coating of Polystyrene Surface with REDV-Gelatin Conjugate for Selective Isolation of Endothelial Cells

2021 ◽  
Vol 904 ◽  
pp. 287-292
Author(s):  
Wan Song Zhang ◽  
Ya Jie Fu ◽  
Rui Wang ◽  
Xuan Hui Qiu ◽  
Ze Yuan Feng ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Function ◽  
Flow Cytometric Analysis ◽  
Internal Surface ◽  
Cost Effective ◽  
Cell Viability Assay ◽  
Simple Method ◽  
Migration Assay ◽  
Gelatin Coating

Endothelial cells (EC), which line the internal surface of blood vessels, play various essential roles in controlling vascular function. The mouse is an important animal model for the study of vascular biology and cardiovascular diseases. However, the isolation of primary EC from the murine aorta is challenging because they are readily contaminated by smooth muscle cells (SMC). A previous study developed a simple method to isolate murine EC from SMC. By taking advantage of the differential sedimentation rate between the two cells, the EC was selectively enriched with collagen-coated polystyrene surfaces. Our study further improved this method by introducing a biomimetic peptide REDV (Arg-Glu-Asp-Val), which may bind specifically to EC but not to SMC or fibroblasts. Firstly, REDV-gelatin conjugate was synthesized by using the amine-to-sulfhydryl crosslinker SMCC. REDV-gelatin coating was then prepared on polystyrene surfaces, and their affinities to EC and SMC were subsequently investigated. Fluorescence microscopy and flow cytometric analysis showed that EC adhesion to the gelatin coating was significantly promoted by REDV peptide conjugation. Moreover, cell migration assay and cell viability assay also showed that the conjugation of REDV does not affect EC migration, and this coating did not show cytotoxicity against EC. This gelatin-REDV coating provides a cost-effective and straightforward tool for isolating EC from SMC, which may facilitate in vitro investigations of EC from mice.

Two-step demineralization of shrimp (Pandalus Borealis) shells using citric acid: An environmentally friendly, safe and cost-effective alternative to the traditional approach

2021 ◽  
Author(s):  
Julia Pohling ◽  
Deepika Dave ◽  
Yi Liu ◽  
Wade Murphy ◽  
Sheila Trenholm
Keyword(s):  
Citric Acid ◽  
Hydrochloric Acid ◽  
Environmental Health ◽  
Traditional Approach ◽  
Health And Safety ◽  
Cost Effective ◽  
Environmental Health And Safety ◽  
Pandalus Borealis ◽  
Chitin Extraction ◽  
Cost Effective Alternative

Removal of minerals from crustacean shells during chitin extraction is traditionally achieved using hydrochloric acid. However, the environmental, health and safety concerns of hydrochloric acid have led to investigation of...

MIC-Drop: A platform for large-scale in vivo CRISPR screens

Science ◽  
2021 ◽  
pp. eabi8870
Author(s):  
Saba Parvez ◽  
Chelsea Herdman ◽  
Manu Beerens ◽  
Korak Chakraborti ◽  
Zachary P. Harmer ◽  
...  
Keyword(s):  
Large Scale ◽  
Cultured Cells ◽  
Cardiac Development ◽  
Droplet Microfluidics ◽  
Model Organisms ◽  
Genetic Screens ◽  
Large Numbers ◽  
And Function ◽  
Genome Scale

CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we report Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we show MIC-Drop can identify small molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discover several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse-genetic screens in model organisms.

An International Campaign for Agricultural and Livestock Genomics (CALG)

2002 ◽  
Vol 06 (24) ◽  
pp. 958-965
Author(s):  
Jun Yu ◽  
Jian Wang ◽  
Huanming Yang
Keyword(s):  
Large Scale ◽  
Cost Effective ◽  
Model Organisms ◽  
Environmental Biology ◽  
Cdna Sequences ◽  
Governmental Agencies ◽  
Technology Innovations ◽  
A Genome ◽  
Starting Point ◽  
The Cost

A coordinated international effort to sequence agricultural and livestock genomes has come to its time. While human genome and genomes of many model organisms (related to human health and basic biological interests) have been sequenced or plugged in the sequencing pipelines, agronomically important crop and livestock genomes have not been given high enough priority. Although we are facing many challenges in policy-making, grant funding, regional task emphasis, research community consensus and technology innovations, many initiatives are being announced and formulated based on the cost-effective and large-scale sequencing procedure, known as whole genome shotgun (WGS) sequencing that produces draft sequences covering a genome from 95 percent to 99 percent. Identified genes from such draft sequences, coupled with other resources, such as molecular markers, large-insert clones and cDNA sequences, provide ample information and tools to further our knowledge in agricultural and environmental biology in the genome era that just comes to its accelerated period. If the campaign succeeds, molecular biologists, geneticists and field biologists from all countries, rich or poor, would be brought to the same starting point and expect another astronomical increase of basic genomic information, ready to convert effectively into knowledge that will ultimately change our lives and environment into a greater and better future. We call upon national and international governmental agencies and organizations as well as research foundations to support this unprecedented movement.

scholarly journals Transfer RNA genes experience exceptionally elevated mutation rates

2018 ◽  
Vol 115 (36) ◽  
pp. 8996-9001 ◽  
Author(s):  
Bryan P. Thornlow ◽  
Josh Hough ◽  
Jacquelyn M. Roger ◽  
Henry Gong ◽  
Todd M. Lowe ◽  
...  
Keyword(s):  
Mutation Rate ◽  
Trna Gene ◽  
Gene Evolution ◽  
Purifying Selection ◽  
Mutation Rates ◽  
Model Organisms ◽  
Biological Synthesis ◽  
Human Populations ◽  
Trna Genes ◽  
Simple Method

Transfer RNAs (tRNAs) are a central component for the biological synthesis of proteins, and they are among the most highly conserved and frequently transcribed genes in all living things. Despite their clear significance for fundamental cellular processes, the forces governing tRNA evolution are poorly understood. We present evidence that transcription-associated mutagenesis and strong purifying selection are key determinants of patterns of sequence variation within and surrounding tRNA genes in humans and diverse model organisms. Remarkably, the mutation rate at broadly expressed cytosolic tRNA loci is likely between 7 and 10 times greater than the nuclear genome average. Furthermore, evolutionary analyses provide strong evidence that tRNA genes, but not their flanking sequences, experience strong purifying selection acting against this elevated mutation rate. We also find a strong correlation between tRNA expression levels and the mutation rates in their immediate flanking regions, suggesting a simple method for estimating individual tRNA gene activity. Collectively, this study illuminates the extreme competing forces in tRNA gene evolution and indicates that mutations at tRNA loci contribute disproportionately to mutational load and have unexplored fitness consequences in human populations.

scholarly journals Energy Sustainability Study of a Rural ICT Telecenter at the Bario Highland

2007 ◽  
Author(s):  
Mohammad Omar Abdullah ◽  
Voon Chun Yung ◽  
Audra Anak Jom ◽  
Alvin Yeo Wee ◽  
Martin Anyi ◽  
...  
Keyword(s):  
Renewable Energy ◽  
Fuel Cell ◽  
Power Systems ◽  
Alternative Energy ◽  
Cost Effective ◽  
Energy Resources ◽  
Successful Implementation ◽  
Combined System ◽  
Pv System ◽  
Simple Method

The eBario project has won the eAsia Award and the Mondialogo Engineering Award in 2004 and 2005 respectively for it’s successful implementation of an Information and Telecommunications Technology Center (ICT) and solar renewable energy-incentive rural community project at the Bario Highland of Sarawak, East Malaysia, Borneo (http://www.unimas.my/ebario/). Although solar photovoltaic (PV) energy has been opted for power generation at the ICT Telecenter for the past five years, there is still a need to investigate the cost-effectiveness of the current energy setup as well as to conduct sustainability study taking into account factors such as system efficiency, weather, costs of fuel, operating costs, as well as to explore the feasibility of implementing alternative energy resources for the rural ICT Telecenter. Recent theoretical study conducted has shown that renewable combined power systems are more sustainable in terms of supplying electricity to the ICT Telecenter, and in a more cost-effective way compared to a standalone PV system which is subject to the cloud and the recent dense haze problems. For that purpose, two combined power systems are being put into consideration namely PV-Hydro and PV-Hydro-Fuel Cell, where the total simulated annualized cost for these two system configurations are US$10,847 and US$76,010 respectively as far as the present location is concerned. The PVHydro-Fuel Cell produces electrical energy at the amount of 3,577 kWh/yr while the annual energy consumption is 3,203 kWhr/yr. On the other hand, PV-Hydro produces 3,789 kWhr/yr of electricity annually load which consumes energy at 3,209 kWhr/yr. Results thus obtained has shown that the PVHydro scheme is expected to have advantages over the existing PV standalone system. Firstly, it is more cost-effective. Secondly, it provides the best outcomes for the local indigenous community and the natural highland environments both for now and the future. Thirdly, it also able to relate the continuity of both economic and social aspects of the local society as a whole. As the combined PV-Hydro system had been chosen, plus for completeness purposes, the present paper also discussed the custom design and construction of a small waterwheel breast-shot hydro-generator, suited to the local location and existing water energy resources. Energy saving design calculations and Sankey diagram showing the energy flows for the new combined system are also given herein. Finally, the energy system performance equations and the performance curves introduced in this study provide a new simple method of evaluating renewable energy systems.

scholarly journals A practical guide to buildde-novoassemblies for single tissues of non-model organisms: the example of a Neotropical frog

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3702 ◽  
Author(s):  
Santiago Montero-Mendieta ◽  
Manfred Grabherr ◽  
Henrik Lantz ◽  
Ignacio De la Riva ◽  
Jennifer A. Leonard ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Cost Effective ◽  
Model Organisms ◽  
Rna Seq ◽  
Assembly Pipeline ◽  
Wide Variability ◽  
History Of ◽  
Inexperienced User

Whole genome sequencing (WGS) is a very valuable resource to understand the evolutionary history of poorly known species. However, in organisms with large genomes, as most amphibians, WGS is still excessively challenging and transcriptome sequencing (RNA-seq) represents a cost-effective tool to explore genome-wide variability. Non-model organisms do not usually have a reference genome and the transcriptome must be assembledde-novo. We used RNA-seq to obtain the transcriptomic profile forOreobates cruralis, a poorly known South American direct-developing frog. In total, 550,871 transcripts were assembled, corresponding to 422,999 putative genes. Of those, we identified 23,500, 37,349, 38,120 and 45,885 genes present in the Pfam, EggNOG, KEGG and GO databases, respectively. Interestingly, our results suggested that genes related to immune system and defense mechanisms are abundant in the transcriptome ofO. cruralis. We also present a pipeline to assist with pre-processing, assembling, evaluating and functionally annotating ade-novotranscriptome from RNA-seq data of non-model organisms. Our pipeline guides the inexperienced user in an intuitive way through all the necessary steps to buildde-novotranscriptome assemblies using readily available software and is freely available at:https://github.com/biomendi/TRANSCRIPTOME-ASSEMBLY-PIPELINE/wiki.

scholarly journals Genome-Scale Screening and Combinatorial Optimization of Gene Overexpression Targets to Improve Cadmium Tolerance in Saccharomyces cerevisiae

2021 ◽  
Vol 12 ◽  
Author(s):  
Yongcan Chen ◽  
Jun Liang ◽  
Zhicong Chen ◽  
Bo Wang ◽  
Tong Si
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Engineering ◽  
Heavy Metal Contamination ◽  
Cadmium Toxicity ◽  
Biomass Accumulation ◽  
Model Organisms ◽  
Cadmium Tolerance ◽  
Wild Type ◽  
Cadmium Resistance ◽  
Genome Scale

Heavy metal contamination is an environmental issue on a global scale. Particularly, cadmium poses substantial threats to crop and human health. Saccharomyces cerevisiae is one of the model organisms to study cadmium toxicity and was recently engineered as a cadmium hyperaccumulator. Therefore, it is desirable to overcome the cadmium sensitivity of S. cerevisiae via genetic engineering for bioremediation applications. Here we performed genome-scale overexpression screening for gene targets conferring cadmium resistance in CEN.PK2-1c, an industrial S. cerevisiae strain. Seven targets were identified, including CAD1 and CUP1 that are known to improve cadmium tolerance, as well as CRS5, NRG1, PPH21, BMH1, and QCR6 that are less studied. In the wild-type strain, cadmium exposure activated gene transcription of CAD1, CRS5, CUP1, and NRG1 and repressed PPH21, as revealed by real-time quantitative PCR analyses. Furthermore, yeast strains that contained two overexpression mutations out of the seven gene targets were constructed. Synergistic improvement in cadmium tolerance was observed with episomal co-expression of CRS5 and CUP1. In the presence of 200 μM cadmium, the most resistant strain overexpressing both CAD1 and NRG1 exhibited a 3.6-fold improvement in biomass accumulation relative to wild type. This work provided a new approach to discover and optimize genetic engineering targets for increasing cadmium resistance in yeast.

scholarly journals Genome-scale metabolic network reconstruction of model animals as a platform for translational research

2021 ◽  
Vol 118 (30) ◽  
pp. e2102344118
Author(s):  
Hao Wang ◽  
Jonathan L. Robinson ◽  
Pinar Kocabas ◽  
Johan Gustafsson ◽  
Mihail Anton ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Metabolic Network ◽  
Model Organisms ◽  
Protein Overexpression ◽  
Sequencing Data ◽  
Proteomics Data ◽  
Gm2 Ganglioside ◽  
Species Specific ◽  
Specific Reactions ◽  
Genome Scale

Genome-scale metabolic models (GEMs) are used extensively for analysis of mechanisms underlying human diseases and metabolic malfunctions. However, the lack of comprehensive and high-quality GEMs for model organisms restricts translational utilization of omics data accumulating from the use of various disease models. Here we present a unified platform of GEMs that covers five major model animals, including Mouse1 (Mus musculus), Rat1 (Rattus norvegicus), Zebrafish1 (Danio rerio), Fruitfly1 (Drosophila melanogaster), and Worm1 (Caenorhabditis elegans). These GEMs represent the most comprehensive coverage of the metabolic network by considering both orthology-based pathways and species-specific reactions. All GEMs can be interactively queried via the accompanying web portal Metabolic Atlas. Specifically, through integrative analysis of Mouse1 with RNA-sequencing data from brain tissues of transgenic mice we identified a coordinated up-regulation of lysosomal GM2 ganglioside and peptide degradation pathways which appears to be a signature metabolic alteration in Alzheimer’s disease (AD) mouse models with a phenotype of amyloid precursor protein overexpression. This metabolic shift was further validated with proteomics data from transgenic mice and cerebrospinal fluid samples from human patients. The elevated lysosomal enzymes thus hold potential to be used as a biomarker for early diagnosis of AD. Taken together, we foresee that this evolving open-source platform will serve as an important resource to facilitate the development of systems medicines and translational biomedical applications.

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