scholarly journals A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila

2019 ◽  
Author(s):  
Fillip Port ◽  
Claudia Strein ◽  
Mona Stricker ◽  
Benedikt Rauscher ◽  
Florian Heigwer ◽  
...  
Keyword(s):  
Large Scale ◽  
Target Genes ◽  
High Efficiency ◽  
Direct Sequencing ◽  
Fine Tuning ◽  
Genetic Screens ◽  
Efficient Gene ◽  
Crispr Screen ◽  
High Gene ◽  
Multicellular Organisms

SUMMARYGenetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1600 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.

scholarly journals A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Fillip Port ◽  
Claudia Strein ◽  
Mona Stricker ◽  
Benedikt Rauscher ◽  
Florian Heigwer ◽  
...  
Keyword(s):  
Large Scale ◽  
Target Genes ◽  
High Efficiency ◽  
Direct Sequencing ◽  
Fine Tuning ◽  
Genetic Screens ◽  
Efficient Gene ◽  
Crispr Screen ◽  
High Gene ◽  
Multicellular Organisms

Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1700 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.

scholarly journals Spectrum of Chemically Induced Mutations From a Large-Scale Reverse-Genetic Screen in Arabidopsis

Genetics ◽  
2003 ◽  
Vol 164 (2) ◽  
pp. 731-740 ◽  
Author(s):  
Elizabeth A Greene ◽  
Christine A Codomo ◽  
Nicholas E Taylor ◽  
Jorja G Henikoff ◽  
Bradley J Till ◽  
...  
Keyword(s):  
Large Scale ◽  
Target Genes ◽  
Nearest Neighbor ◽  
Large Data ◽  
Chemical Mutagenesis ◽  
Reverse Genetic ◽  
Genetic Screens ◽  
Induced Mutations ◽  
Data Set ◽  
Chemically Induced

AbstractChemical mutagenesis has been the workhorse of traditional genetics, but it has not been possible to determine underlying rates or distributions of mutations from phenotypic screens. However, reverse-genetic screens can be used to provide an unbiased ascertainment of mutation statistics. Here we report a comprehensive analysis of ∼1900 ethyl methanesulfonate (EMS)-induced mutations in 192 Arabidopsis thaliana target genes from a large-scale TILLING reverse-genetic project, about two orders of magnitude larger than previous such efforts. From this large data set, we are able to draw strong inferences about the occurrence and randomness of chemically induced mutations. We provide evidence that we have detected the large majority of mutations in the regions screened and confirm the robustness of the high-throughput TILLING method; therefore, any deviations from randomness can be attributed to selectional or mutational biases. Overall, we detect twice as many heterozygotes as homozygotes, as expected; however, for mutations that are predicted to truncate an encoded protein, we detect a ratio of 3.6:1, indicating selection against homozygous deleterious mutations. As expected for alkylation of guanine by EMS, >99% of mutations are G/C-to-A/T transitions. A nearest-neighbor bias around the mutated base pair suggests that mismatch repair counteracts alkylation damage.

scholarly journals Engineering digitizer circuits for chemical and genetic screens in human cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nicole M. Wong ◽  
Elizabeth Frias ◽  
Frederic D. Sigoillot ◽  
Justin H. Letendre ◽  
Marc Hild ◽  
...  
Keyword(s):  
Large Scale ◽  
Dynamic Range ◽  
Genetic Screens ◽  
Genetic Circuits ◽  
Transient Responses ◽  
Reporter Activity ◽  
Genome Wide ◽  
Pathway Activation ◽  
Crispr Screen ◽  
Or Genes

AbstractCell-based transcriptional reporters are invaluable in high-throughput compound and CRISPR screens for identifying compounds or genes that can impact a pathway of interest. However, many transcriptional reporters have weak activities and transient responses. This can result in overlooking therapeutic targets and compounds that are difficult to detect, necessitating the resource-consuming process of running multiple screens at various timepoints. Here, we present RADAR, a digitizer circuit for amplifying reporter activity and retaining memory of pathway activation. Reporting on the AP-1 pathway, our circuit identifies compounds with known activity against PKC-related pathways and shows an enhanced dynamic range with improved sensitivity compared to a classical reporter in compound screens. In the first genome-wide pooled CRISPR screen for the AP-1 pathway, RADAR identifies canonical genes from the MAPK and PKC pathways, as well as non-canonical regulators. Thus, our scalable system highlights the benefit and versatility of using genetic circuits in large-scale cell-based screening.

scholarly journals In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yu Wang ◽  
Haijiao Cheng ◽  
Yang Liu ◽  
Ye Liu ◽  
Xiao Wen ◽  
...  
Keyword(s):  
Large Scale ◽  
High Efficiency ◽  
Metabolic Reprogramming ◽  
Fine Tuning ◽  
Ex Situ ◽  
Library Size ◽  
Base Editing ◽  
Large Numbers ◽  
Multigene Expression

AbstractReprogramming complex cellular metabolism requires simultaneous regulation of multigene expression. Ex-situ cloning-based methods are commonly used, but the target gene number and combinatorial library size are severely limited by cloning and transformation efficiencies. In-situ methods such as multiplex automated genome engineering (MAGE) depends on high-efficiency transformation and incorporation of heterologous DNA donors, which are limited to few microorganisms. Here, we describe a Base Editor-Targeted and Template-free Expression Regulation (BETTER) method for simultaneously diversifying multigene expression. BETTER repurposes CRISPR-guided base editors and in-situ generates large numbers of genetic combinations of diverse ribosome binding sites, 5’ untranslated regions, or promoters, without library construction, transformation, and incorporation of DNA donors. We apply BETTER to simultaneously regulate expression of up to ten genes in industrial and model microorganisms Corynebacterium glutamicum and Bacillus subtilis. Variants with improved xylose catabolism, glycerol catabolism, or lycopene biosynthesis are respectively obtained. This technology will be useful for large-scale fine-tuning of multigene expression in both genetically tractable and intractable microorganisms.

The Potential of Photoelectrochemical Carbon Sinks

2018 ◽  
Author(s):  
Matthias May ◽  
Kira Rehfeld
Keyword(s):  
Global Warming ◽  
High Temperature ◽  
Greenhouse Gas ◽  
Large Scale ◽  
High Efficiency ◽  
Carbon Sink ◽  
Solar Fuels ◽  
Negative Emissions ◽  
Viable Approach ◽  
Low Sensitivity

Greenhouse gas emissions must be cut to limit global warming to 1.5-2C above preindustrial levels. Yet the rate of decarbonisation is currently too low to achieve this. Policy-relevant scenarios therefore rely on the permanent removal of CO<sub>2</sub> from the atmosphere. However, none of the envisaged technologies has demonstrated scalability to the decarbonization targets for the year 2050. In this analysis, we show that artificial photosynthesis for CO<sub>2</sub> reduction may deliver an efficient large-scale carbon sink. This technology is mainly developed towards solar fuels and its potential for negative emissions has been largely overlooked. With high efficiency and low sensitivity to high temperature and illumination conditions, it could, if developed towards a mature technology, present a viable approach to fill the gap in the negative emissions budget.<br>

The Potential of Photoelectrochemical Carbon Sinks

2018 ◽  
Author(s):  
Matthias May ◽  
Kira Rehfeld
Keyword(s):  
Global Warming ◽  
High Temperature ◽  
Greenhouse Gas ◽  
Large Scale ◽  
High Efficiency ◽  
Carbon Sink ◽  
Solar Fuels ◽  
Negative Emissions ◽  
Viable Approach ◽  
Low Sensitivity

Greenhouse gas emissions must be cut to limit global warming to 1.5-2C above preindustrial levels. Yet the rate of decarbonisation is currently too low to achieve this. Policy-relevant scenarios therefore rely on the permanent removal of CO<sub>2</sub> from the atmosphere. However, none of the envisaged technologies has demonstrated scalability to the decarbonization targets for the year 2050. In this analysis, we show that artificial photosynthesis for CO<sub>2</sub> reduction may deliver an efficient large-scale carbon sink. This technology is mainly developed towards solar fuels and its potential for negative emissions has been largely overlooked. With high efficiency and low sensitivity to high temperature and illumination conditions, it could, if developed towards a mature technology, present a viable approach to fill the gap in the negative emissions budget.<br>

Peculiarities of the phytosanitary state of pome fruits in industrial horticulture

2020 ◽  
Vol 60 (1) ◽  
pp. 159-168
Author(s):  
V. V. Antonenko ◽  
A. V. Zubkov ◽  
S. N. Kruchina
Keyword(s):  
Mechanism Of Action ◽  
Large Scale ◽  
High Efficiency ◽  
Fruit Trees ◽  
Fruit Crops ◽  
Planting Material ◽  
Relative Safety ◽  
Pome Fruits ◽  
Weed Plants ◽  
Spread Of Infection

Data were obtained on the basis of the results of research carried out on the territory of the educational and experimental farm of the Timiryazev State Agrarian University, in Moscow during 2018-2019. As a result of the surveys, the most dangerous diseases and pests of pome crops on the territory of this farm were established. The most resistant apple and pear varieties to major diseases have been identified. Peculiarities of development of alternariosis on pear are described, the harmfulness of the disease on pear and apple seedlings is noted. A possible role in the transfer of alternariosis infection from garden-protective plantations and weed vegetation to fruit trees was noted. A possible role has been established in the transport of septoriosis, powdery dew infection from dicotyledonous weeds plants. The peculiarities of the spread of infection under the influence of wind direction are noted. The results and peculiarities of the application of various methods of scaring birds in the orchard are presented. As a result of route surveys the most harmful weed plants have been identified. The possibility of using herbicides of different mechanism of action in fruit gardens for weed control has been studied. High efficiency and relative safety of application of herbicides of contact action in nursery fields, operational orchards and for control of piglets on fruit trees are shown. Recommendations are given for the use of soil and systemic herbicides of soil in seedlings beds, the first and second fields of the nursery, as well as in the process of production of large-scale planting material and operational orchards of fruit crops. The safety of the herbicides in question is established when used in accordance with the recommended methods of use.

scholarly journals Functionalization of Congo red dye as a light harvester on solar cell

2020 ◽  
Vol 18 (1) ◽  
pp. 287-294
Author(s):  
Harsasi Setyawati ◽  
Handoko Darmokoesoemo ◽  
Irmina Kris Murwani ◽  
Ahmadi Jaya Permana ◽  
Faidur Rochman
Keyword(s):  
Solar Cell ◽  
Congo Red ◽  
Large Scale ◽  
High Efficiency ◽  
Solar Cell Device ◽  
Dye Sensitized ◽  
Congo Red Dye ◽  
Elemental Analyses ◽  
Red Dye ◽  
Solar Cell Technology

AbstractThe demands of ecofriendly technologies to produce a reliable supply of renewable energy on a large scale remains a challenge. A solar cell based on DSSC (Dye-Sensitized Solar Cell) technology is environmentally friendly and holds the promise of a high efficiency in converting sunlight into electricity. This manuscript describes the development of a light harvester system as a main part of a DSSC. Congo red dye has been functionalized with metals (Fe, Co, Ni), forming a series of complexes that serve as a novel light harvester on the solar cell. Metal-congo red complexes have been characterized by UV-VIS and FTIR spectroscopy, and elemental analyses. The performance of metal complexes in capturing photons from sunlight has been investigated in a solar cell device. The incorporation of metals to congo red successfully improved of the congo red efficiency as follows: Fe(II)-congo red, Co(II)-congo red and Ni(II)-congo red had efficiencies of 8.17%, 6.13% and 2.65%, respectively. This research also discusses the effect of metal ions on the ability of congo red to capture energy from sunlight.

scholarly journals A low-area high-efficiency video coding inverse transform core using resource and time sharing architecture

2020 ◽  
Vol 2020 (1) ◽  
Author(s):  
Yuan-Ho Chen ◽  
Chieh-Yang Liu
Keyword(s):  
Video Coding ◽  
Large Scale ◽  
High Efficiency ◽  
Vlsi Design ◽  
Cmos Technology ◽  
Oxide Semiconductor ◽  
Time Sharing ◽  
High Efficiency Video Coding ◽  
Low Area ◽  
Matrix Products

AbstractIn this paper, a very-large-scale integration (VLSI) design that can support high-efficiency video coding inverse discrete cosine transform (IDCT) for multiple transform sizes is proposed. The proposed two-dimensional (2-D) IDCT is implemented at a low area by using a single one-dimensional (1-D) IDCT core with a transpose memory. The proposed 1-D IDCT core decomposes a 32-point transform into 16-, 8-, and 4-point matrix products according to the symmetric property of the transform coefficient. Moreover, we use the shift-and-add unit to share hardware resources between multiple transform dimension matrix products. The 1-D IDCT core can simultaneously calculate the first- and second-dimensional data. The results indicate that the proposed 2-D IDCT core has a throughput rate of 250 MP/s, with only 110 K gate counts when implemented into the Taiwan semiconductor manufacturing (TSMC) 90-nm complementary metal-oxide-semiconductor (CMOS) technology. The results show the proposed circuit has the smallest area supporting the multiple transform sizes.

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