scholarly journals Rapid generation of homozygous fluorescent knock-in human cells using CRISPR/Cas9 genome editing and validation by automated imaging and digital PCR screening

2021 ◽  
Author(s):  
Moritz Kueblbeck ◽  
Andrea Callegari ◽  
Beatriz Serrano-Solano ◽  
Jan Ellenberg
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Single Cells ◽  
Pcr Primers ◽  
Digital Pcr ◽  
Pcr Screening ◽  
Single Genome ◽  
Rapid Generation ◽  
Fluorescent Tags ◽  
High Throughput Assay

We have previously described a protocol for genome engineering of mammalian cultured cancer cells with CRISPR/Cas9 to generate homozygous knock-ins of fluorescent tags into endogenous genes. Here, we are updating this protocol to reflect major improvements in the workflow regarding efficiency and throughput. In brief, we have improved our method by combining high efficiency electroporation of optimized CRISPR/Cas9 reagents, screening of single cell derived clones by automated bright field and fluorescence imaging, rapidly assessing the number of tagged alleles and potential off-targets using digital PCR (dPCR) and automated data analysis. Compared to the original protocol, our current procedure (i) significantly increases the efficiency of tag integration, (ii) automates the identification of clones derived from single cells with correct subcellular localization of the tagged protein and (iii) provides a quantitative and high throughput assay to measure the number of on- and off-target integrations with dPCR. The increased efficiency of the new procedure reduces the number of clones that need to be analysed in-depth by more than ten-fold, and yields up to 20% of homozygous clones in polyploid cancer cell lines in a single genome engineering round. Overall, we were able to dramatically reduce the hands-on time from 30 days to 10 days during the overall ~10 weeks procedure, allowing a single person to process up to 5 genes in parallel, assuming that validated reagents - e.g. PCR-primers, dPCR-assays, Western Blot antibodies - are available.

scholarly journals Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR

2021 ◽  
Author(s):  
Christian Schulze ◽  
Anne-Catrin Geuthner ◽  
Dietrich Mäde
Keyword(s):  
Durum Wheat ◽  
Real Time ◽  
Bread Wheat ◽  
Common Wheat ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Food Fraud ◽  
Single Genome ◽  
Cereal Species

AbstractFood fraud is becoming a prominent topic in the food industry. Thus, valid methods for detecting potential adulterations are necessary to identify instances of food fraud in cereal products, a significant component of human diet. In this work, primer–probe systems for real-time PCR and droplet digital PCR (ddPCR) for the detection of these cereal species: bread wheat (together with spelt), durum wheat, rye and barley for real-time PCR and ddPCR were established, optimized and validated. In addition, it was projected to validate a molecular system for differentiation of bread wheat and spelt; however, attempts for molecular differentiation between common wheat and spelt based on the gene GAG56D failed because of the genetic variability of the molecular target. Primer–probe systems were further developed and optimized on the basis of alignments of DNA sequences, as well as already developed PCR systems. The specificity of each system was demonstrated on 10 (spelt), 11 (durum wheat and rye) and 12 (bread wheat) reference samples. Specificity of the barley system was already proved in previous work. The calculated limits of detection (LOD95%) were between 2.43 and 4.07 single genome copies in real-time PCR. Based on the “three droplet rule”, the LOD95% in ddPCR was calculated to be 9.07–13.26 single genome copies. The systems were tested in mixtures of flours (rye and common wheat) and of semolina (durum and common wheat). The methods proved to be robust with regard to the tested conditions in the ddPCR. The developed primer–probe systems for ddPCR proved to be effective in quantitatively detecting the investigated cereal species rye and common wheat in mixtures by taking into account the haploid genome weight and the degree of milling of a flour. This method can correctly detect proportions of 50%, 60% and 90% wholemeal rye flour in a mixture of wholemeal common wheat flour. Quantitative results depend on the DNA content, on ploidy of cereal species and are also influenced by comminution. Hence, the proportion of less processed rye is overestimated in higher processed bread wheat and adulteration of durum wheat by common wheat by 1–5% resulted in underestimation of common wheat.

Testing of solid oxide cells at high current densities

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
André Weber
Keyword(s):  
Single Cell ◽  
High Performance ◽  
High Efficiency ◽  
Electrochemical Impedance ◽  
Single Cells ◽  
Operating Conditions ◽  
Solid Oxide ◽  
Current Densities ◽  
Cell Testing ◽  
The Impact

Abstract Solid Oxide Cells (SOCs) have gained an increasing interest as electrochemical energy converters due to their high efficiency, fuel flexibility and ability of reversible fuel cell/electrolysis operation. During the development process as well as in quality assurance tests, the performance of single cells and cell stacks is commonly evaluated by means of current/voltage- (CV-) characteristics. Despite of the fact that the measurement of a CV-characteristic seems to be simple compared to more complex, dynamic methods as electrochemical impedance spectroscopy or current interrupt techniques, the resulting performance strongly depends on the test setup and the chosen operating conditions. In this paper, the impact of different single cell testing environments and operating conditions on the CV-characteristic of high performance cells is discussed. The influence of cell size, contacting and current collection, contact pressure, fuel flow rate and composition on the achievable cell performance is presented and limitations arising from the test bed and testing conditions will be pointed out. As today’s high performance cells are capable of delivering current densities of several ampere per cm2 a special emphasis will be laid on single cell testing in this current range.

Design Enhancements for High Performance Dye-Sensitized Solar Cells

2013 ◽  
Vol 135 (3) ◽  
Author(s):  
K. Parmar ◽  
A. Kianimanesh ◽  
T. Freiheit ◽  
S. S. Park
Keyword(s):  
Solar Cells ◽  
High Performance ◽  
Fossil Fuels ◽  
High Efficiency ◽  
Single Cells ◽  
Incident Light ◽  
Dye Sensitized Solar Cells ◽  
Rear Side ◽  
Dye Sensitized ◽  
Fresnel Lenses

Due to the abundance of solar energy, solar cells are considered as a renewable source of energy to replace conventional fossil fuels. Compared to the silicon-based photovoltaic (PV) cell, the next generation dye-sensitized solar cell (DSSC) offers the advantages of increased absorption of visible light, high efficiency potential, less energy intensive and lower-cost manufacturing process, colorable design, and lightweight material options. DSSC is a photo-electrochemical system that is based on a photosensitive dye-sensitized semiconductor (mostly titanium dioxide, TiO2) anode and an iodide-based electrolyte. In order to improve the performance of current DSSC systems, we proposed various design improvement schemes through the use of TiO2 nanotube (TONT) arrays and a multistack design of single cells. Through design modifications, approximately 38% improvement in the performance compared to conventional DSSC is reported. Moreover, optical enhancements to increase the amount of incident light on the cell were applied to DSSCs to further improve its performance by application of Fresnel lenses on top of the DSSC and the use of light reflecting material such as Aluminum on the rear side of the cell. The polarization curves for different designs were measured using a potentiostat and the performance of each cell was compared. Optical enhancements improved the power output by 27% compared to normal cells. A semi-empirical DSSC model was also developed based on the experimental results and the change in the performance of different designs was examined. Based on the model, the necessary conditions for maximum performance could be determined.

scholarly journals High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

2021 ◽  
Author(s):  
Jongmin Woo ◽  
Sarah M. Williams ◽  
Victor Aguilera-Vazquez ◽  
Ryan L. Sontag ◽  
Ronald J. Moore ◽  
...  
Keyword(s):  
Single Cell ◽  
High Efficiency ◽  
Single Cells ◽  
Protein Recovery ◽  
Processing Capacity ◽  
Protein Abundance ◽  
Proteome Coverage ◽  
Low Protein ◽  
Functional Differences ◽  
Cell Proteome

AbstractGlobal quantification of protein abundances in single cells would provide more direct information on cellular function phenotypes and complement transcriptomics measurements. However, single-cell proteomics (scProteomics) is still immature and confronts technical challenges, including limited proteome coverage, poor reproducibility, as well as low throughput. Here we describe a nested nanoPOTS (N2) chip to dramatically improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip allows reducing cell digestion volume to <30 nL and increasing processing capacity to > 240 single cells in one microchip. In the analysis of ∼100 individual cells from three different cell lines, we demonstrate the N2 chip-based scProteomics platform can robustly quantify ∼1500 proteins and reveal functional differences. Our analysis also reveals low protein abundance variations (median CVs < 16.3%), highlighting the utility of such measurements, and also suggesting the single-cell proteome is highly stable for the cells cultured under identical conditions.

scholarly journals First Report of CRISPR/Cas9 Gene Editing in Castanea sativa Mill

2021 ◽  
Vol 12 ◽  
Author(s):  
Vera Pavese ◽  
Andrea Moglia ◽  
Elena Corredoira ◽  
Mª Teresa Martínez ◽  
Daniela Torello Marinoni ◽  
...  
Keyword(s):  
Somatic Embryos ◽  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Chlorophyll Biosynthesis ◽  
Point Mutations ◽  
Castanea Sativa ◽  
Visual Assessment ◽  
European Chestnut ◽  
Selection Medium

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored in forest species. In this study, we reported the first example of CRISPR/Cas9-mediated gene editing in Castanea genus. As a proof of concept, we targeted the gene encoding phytoene desaturase (pds), whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Globular and early torpedo-stage somatic embryos of Castanea sativa (European chestnut) were cocultured for 5 days with a CRISPR/Cas9 construct targeting two conserved gene regions of pds and subsequently cultured on a selection medium with kanamycin. After 8 weeks of subculture on selection medium, four kanamycin-resistant embryogenetic lines were isolated. Genotyping of these lines through target Sanger sequencing of amplicons revealed successful gene editing. Cotyledonary somatic embryos were maturated on maltose 3% and cold-stored at 4°C for 2 months. Subsequently, embryos were subjected to the germination process to produce albino plants. This study opens the way to the use of the CRISPR/Cas9 system in European chestnut for biotechnological applications

scholarly journals Sample Pooling as an efficient strategy for SARS-COV-2 RT-PCR screening: a multicenter study in Spain

2020 ◽  
Author(s):  
Adolfo de Salazar ◽  
Antonio Aguilera ◽  
Rocio Trastoy ◽  
Ana Fuentes ◽  
Juan Carlos Alados ◽  
...  
Keyword(s):  
High Efficiency ◽  
Rna Extraction ◽  
Dilution Effect ◽  
Acid Extraction ◽  
Predictive Values ◽  
Pcr Screening ◽  
Prospective Multicentre Study ◽  
Clinical Laboratories ◽  
Sample Pooling ◽  
Sensitivity Specificity

Importance: The actual demand on SARS-CoV-2 diagnosis is a current challenge for clinical laboratories. Sample pooling may help to ameliorate workload in clinical laboratories. Objective: to evaluate the efficacy of sample pooling compared to the individual analysis for the diagnosis of CoVID-19, by using different commercial platforms for nucleic acid extraction and amplification. Design and settings: observational, prospective, multicentre study across 9 Spanish clinical microbiology laboratories including SARS-CoV-2 RNA testing performed in April 2020, during the first three days after acceptance to participate. Participants and Methods: 3519 naso-oro-pharyngeal samples received at the participating laboratories were processed individually and in pools (351 pools) according to the existing methodology in each of the centres. Results: We found that 253 pools (2519 samples) were negative, and 99 pools (990 samples) were positive; with 241 positive samples (6.85%), our pooling strategy would have saved 2167 PCR tests. For 29 pools (made out of 290 samples) we found discordant results when compared to their correspondent individual samples: in 24/29 pools (30 samples), minor discordances were found; for five pools (5 samples), we found major discordances. Sensitivity, specificity, positive and negative predictive values for pooling were 97.93%, 100%, 100% and 99.85% respectively; accuracy was 99.86% and kappa concordant coefficient was 0.988. As a result of the sample dilution effect of pooling, a loss of 2-3 Cts was observed for E, N or RdRP genes. Conclusion: we show a high efficiency of pooling strategies for SARS-CoV-2 RNA testing, across different RNA extraction and amplification platforms, with excellent performance in terms of sensitivity, specificity, and positive and negative predictive values. We believe that our results may help clinical laboratories to respond to the actual demand and clinical need on SARS-CoV-2 testing, especially for the screening of low prevalence populations.

scholarly journals Efficient targeted integration directed by short homology in zebrafish and mammalian cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Wesley A Wierson ◽  
Jordan M Welker ◽  
Maira P Almeida ◽  
Carla M Mann ◽  
Dennis A Webster ◽  
...  
Keyword(s):  
High Frequency ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
High Efficiency ◽  
Cost Effective ◽  
Strand Break ◽  
End Joining ◽  
Cost Effective Approach ◽  
Targeted Integration

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

Synthesis and Characterization of Nanocrystalline La0.65Sr0.3 MnO3 and La0.8Sr0.2MnO3 Cathode Powders by Auto-ignition Technique for Solid Oxide Fuel Cells (SOFC)

2016 ◽  
Vol 19 (2) ◽  
pp. 065-076
Author(s):  
G. N. Almutairi ◽  
M. Ghouse ◽  
Y. M. Alyousef ◽  
F. S. Alenazey
Keyword(s):  
Fuel Cells ◽  
Solid Oxide Fuel Cells ◽  
High Efficiency ◽  
Single Cells ◽  
Average Crystallite Size ◽  
Solid Oxide ◽  
Oxide Fuel ◽  
Functional Layer ◽  
Auto Combustion

Solid Oxide fuel Cells (SOFCs) are considered to be one of the most promising energy conversion devices that have several advantages such as high efficiency, system compactness and low environmental pollution. In the present investigation La0.65Sr0.3MnO3 (LSM-1) and La0.8Sr0.2MnO3 (LSM-2) nanoceramic powders were prepared by citrate-nitrate route of auto-combustion with citrate to nitrate (c/n) ratio 0.50 to see the effect of these cathode powders on the performance of SOFC cells. The as prepared powder were calcined at 900oC for 4hrs using the Thermolyne 47900 furnace to remove carboneous residues and characterized them using SEM / EDS, XRD, TGA techniques and their results are presented . From calculations using Debye Scherrer’s equation, the average crystallite size of the powders were found to be around 16nm. The SEM indicates the particle sizes are within the range of around 200nm.The surface area of the calcined LSM-2 powder was found to be ~21m2/g. The TGA studies indicate the completion of combustion since there was no further weight loss after reaching temperature of ~ 650oC. Also, Electrochemical characterization of LSM cathode powders were carried out by coating these powders (as cathode functional layer CFL-Bottom and current collector layer CL- Top) using Screen printing on the SOFC half cells (NiO-YSZ+YSZ) procured from CGCRI, Kolkata, India with a cell size of 16mm dia x1.6mm and tested these cells with H2-O2 at 750-800oC with the flow rates of 100-200 sccm. The results of the performances of single cells are presented in this paper. The Current density and powder density values obtained are 0.80A/cm2 (at 0.7V) and 0.55 W/cm2 at 800oC with 200 sccm of hydrogen and oxygen respectively. The area surface resistance (ASR) values obtained were ~0.50 Ωcm2 at 0.7V at 800oC.

scholarly journals Co-incident insertion enables high efficiency genome engineering in mouse embryonic stem cells

2016 ◽  
Vol 44 (16) ◽  
pp. 7997-8010 ◽  
Author(s):  
Brian R. Shy ◽  
Matthew S. MacDougall ◽  
Ryan Clarke ◽  
Bradley J. Merrill
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Genome Engineering ◽  
High Efficiency ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells

Hydrodynamic shuttling for deterministic high-efficiency multiple single-cell capture in a microfluidic chip

Lab on a Chip ◽  
2019 ◽  
Vol 19 (8) ◽  
pp. 1370-1377 ◽  
Author(s):  
Cheng-Kun He ◽  
Ya-Wen Chen ◽  
Ssu-Han Wang ◽  
Chia-Hsien Hsu
Keyword(s):  
Single Cell ◽  
Biomedical Research ◽  
Microfluidic Chip ◽  
High Efficiency ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Cell Capture

A new microfluidics technique for high-efficiency paring and analyzing multiple single cells can facilitate cellular heterogeneity studies important for biological and biomedical research.

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