scholarly journals Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Yu Hisano ◽  
Tetsushi Sakuma ◽  
Shota Nakade ◽  
Rie Ohga ◽  
Satoshi Ota ◽  
...  
Keyword(s):  
High Efficiency ◽  
Model Organisms ◽  
Cleavage Sites ◽  
Simple Method ◽  
Exogenous Dna ◽  
Dna Integration ◽  
Homologous Sequences ◽  
Genome Modification ◽  
Vector Sequences ◽  
The Right

Abstract The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10–40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.

scholarly journals CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Rhoda Mae C. Simora ◽  
De Xing ◽  
Max R. Bangs ◽  
Wenwen Wang ◽  
Xiaoli Ma ◽  
...  
Keyword(s):  
Ictalurus Punctatus ◽  
Channel Catfish ◽  
High Efficiency ◽  
Gene Knockout ◽  
Chromosome 1 ◽  
Model Organisms ◽  
Coding Region ◽  
Exogenous Dna ◽  
Efficient System ◽  
Gene Integration

AbstractCRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.

scholarly journals Agrobacterium-Mediated Transformation of Aspergillus awamori in the Absence of Full-Length VirD2, VirC2, or VirE2 Leads to Insertion of Aberrant T-DNA Structures

2004 ◽  
Vol 186 (7) ◽  
pp. 2038-2045 ◽  
Author(s):  
Caroline B. Michielse ◽  
Arthur F. J. Ram ◽  
Paul J. J. Hooykaas ◽  
Cees A. M. J. J. van den Hondel
Keyword(s):  
Single Copy ◽  
Full Length ◽  
Aspergillus Awamori ◽  
Wild Type ◽  
Dna Structures ◽  
Dna Integration ◽  
Virulence Proteins ◽  
Type Transformation ◽  
The Right ◽  
Transposon Insertions

ABSTRACT Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.

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 Heterogeneous Catalytic Synthesis of 2-Methylbenzimidazole from 2-Nitroaniline and Ethanol Over Mg Modified Cu-Pd/γ-Al2O3

Catalysts ◽  
2018 ◽  
Vol 9 (1) ◽  
pp. 8 ◽  
Author(s):  
Feng Feng ◽  
Yaqin Deng ◽  
Zheng Cheng ◽  
Xiaoliang Xu ◽  
Qunfeng Zhang ◽  
...  
Keyword(s):  
Active Sites ◽  
Hydrogen Transfer ◽  
High Efficiency ◽  
Simple Procedure ◽  
Direct Synthesis ◽  
Reaction System ◽  
Simple Method ◽  
Heterogeneous Catalytic ◽  
Coupled Reaction ◽  
Al2o3 Catalyst

The direct synthesis of benzimidazoles from 2-nitroaniline and ethanol over Cu-Pd/γ-Al2O3 catalysts has the advantages of requiring easily available starting materials, having high efficiency, and a simple procedure. The modification by Mg of the Cu-Pd/γ-Al2O3 catalyst could improve the catalytic activity significantly. The addition of Mg to the Cu-Pd/γ-Al2O3 catalyst could maintain and promote the formation of CuPd alloy active sites. Meanwhile, the basicity of the support was enhanced appropriately by Mg, which generated more basic sites (Al-Oδ−) to accelerate the dehydrogenation of alcohol and increased the rate of the whole coupled reaction. The 2-nitroaniline was completely converted over Cu-Pd/(Mg)γ-Al2O3 after reacting for six hours, and the yield of 2-methylbenzimidazole was 98.8%. The results of this work provide a simple method to develop a more efficient catalyst for the “alcohol-dehydrogenation, hydrogen transfer and hydrogenation” coupled reaction system.

scholarly journals Identification of high-efficiency 3′GG gRNA motifs in indexed FASTA files with ngg2

2015 ◽  
Vol 1 ◽  
pp. e33 ◽  
Author(s):  
Elisha D. Roberson
Keyword(s):  
High Efficiency ◽  
Homo Sapiens ◽  
Model Organisms ◽  
Proof Of Concept ◽  
Protein Coding ◽  
C Elegans ◽  
Protein Coding Genes ◽  
Starting Point ◽  
Command Line Tool ◽  
Reference Genomes

CRISPR/Cas9 is emerging as one of the most-used methods of genome modification in organisms ranging from bacteria to human cells. However, the efficiency of editing varies tremendously site-to-site. A recent report identified a novel motif, called the 3′GG motif, which substantially increases the efficiency of editing at all sites tested inC. elegans. Furthermore, they highlighted that previously published gRNAs with high editing efficiency also had this motif. I designed a Python command-line tool, ngg2, to identify 3′GG gRNA sites from indexed FASTA files. As a proof-of-concept, I screened for these motifs in six model genomes:Saccharomyces cerevisiae,Caenorhabditis elegans,Drosophila melanogaster,Danio rerio,Mus musculus, andHomo sapiens. I also scanned the genomes of pig (Sus scrofa) and African elephant (Loxodonta africana) to demonstrate the utility in non-model organisms. I identified more than 60 million single match 3′GG motifs in these genomes. Greater than 61% of all protein coding genes in the reference genomes had at least one unique 3′GG gRNA site overlapping an exon. In particular, more than 96% of mouse and 93% of human protein coding genes have at least one unique, overlapping 3′GG gRNA. These identified sites can be used as a starting point in gRNA selection, and the ngg2 tool provides an important ability to identify 3′GG editing sites in any species with an available genome sequence.

scholarly journals Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

2019 ◽  
Author(s):  
Remi L. Gratacap ◽  
Tim Regan ◽  
Carola E. Dehler ◽  
Samuel A.M. Martin ◽  
Pierre Boudinot ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Cell Line ◽  
Genome Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Genetic Resistance ◽  
Model Organisms ◽  
Fish Cell ◽  
Genome Wide ◽  
Lentivirus Transduction

1AbstractGenome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques. In the current study, an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214) was developed. As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated. The optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.

scholarly journals A high-efficiency and ultrathin transmission-type circular polarization converter based on surface structure

2021 ◽  
Vol 8 ◽  
pp. 4
Author(s):  
Peng Xu ◽  
Wei Xiang Jiang ◽  
Xiao Cai ◽  
Yue Gou ◽  
Tie Jun Cui
Keyword(s):  
High Efficiency ◽  
Transmission Mode ◽  
Polarization Converter ◽  
Periodic Surface ◽  
Circularly Polarized ◽  
Left Handed ◽  
Via Holes ◽  
Working Frequency ◽  
The Right ◽  
Good Agreement

In this paper, we propose, design and fabricate a kind of ultrathin and high-efficiency circularly polarization converter based on artificially engineered surfaces in the transmission mode. The converter is composed of double-layer periodic surface structures with cross slots. The top and bottom layers are printed on both sides of the F4B substrate and connected by metallic via holes. The proposed converter can transform the right-handed circularly polarized incident electromagnetic (EM) wave to a left-handed circularly-polarized one with near-unity efficiency in the transmission mode, or vice versa. We explain the conversion mechanism based on numerical simulations and equivalent circuit (EC) theory. The measured result has a good agreement with the simulated one in the working frequency band. Such ultrathin polarization converters can be used in wireless microwave communication, remote sensing, and EM imaging where circularly polarization diversity is needed.

Five Strip-Type Dye Sensitized Solar Cells with Metal Grid Lines

2007 ◽  
Vol 119 ◽  
pp. 315-318
Author(s):  
Won Jae Lee ◽  
Easwaramoorthi Ramasamy ◽  
Dong Yoon Lee ◽  
Jae Sung Song
Keyword(s):  
Solar Cells ◽  
High Efficiency ◽  
Dye Sensitized Solar Cells ◽  
Standard Condition ◽  
Light Condition ◽  
Fluorescent Light ◽  
Simple Method ◽  
Dye Sensitized ◽  
Large Size ◽  
Sensitized Solar Cells

Dye sensitized solar cells (DSSC) have great potential alternative to expensive conventional solar cells, since high efficiency and relatively simple fabrication process. However, in large size cell, there is a key factor that delayed the entry of such cells in commercial market. Performance of large size cell is lower than small size cells, since a carrier loss occurs in high resistive TCO glass substrate. Here we demonstrate a simple method to reduce resistive loss and efficient collection of photo generated carriers via strip type cells with metal grids. Using strip type cells, we constitute series and parallel type DSSC panels in order to achieve required voltage and current respectively. Stripe cells were prepared from commercial TiO2 powder by screen printing method. In addition, metal grids were established adjacent to sealant line. Using these as unit cell, portable DSSC panels were assembled and I-V performance was carried out in indoor light condition (fluorescent light, 30mW/cm2) and standard condition (Pin 100 mW/cm2, AM 1.5).

Evaluation of the Service Performance of Ships

2005 ◽  
Vol 42 (04) ◽  
pp. 177-183
Author(s):  
Poul Andersen ◽  
Anne-Sophie Borrod ◽  
Hervé Blanchot
Keyword(s):  
Input Data ◽  
High Efficiency ◽  
Service Performance ◽  
The Other ◽  
Simple Method ◽  
Propulsive Efficiency ◽  
Order Of Magnitude ◽  
Time Periods

A simple method has been established for the evaluation of the service performance of ships. Input data are easily collected daily on board and transformed to a well-defined condition that makes possible the comparison between ships, for instance, sister ships, and between different time periods of voyages for the same ship. The procedure has been applied to two ships that are identical, with the only exception that one has a conventional propeller, whereas the other one is fitted with a high-efficiency propeller of the KAPPEL type. The results are obtained from a period of 2 years steaming for both vessels. They clearly confirm the increase of propulsive efficiency obtained with the KAPPEL propeller in the order of magnitude of 4%.

scholarly journals CRISPR/Cas9 for Cancer Therapy: Hopes and Challenges

Biomedicines ◽  
2018 ◽  
Vol 6 (4) ◽  
pp. 105 ◽  
Author(s):  
Marta Martinez-Lage ◽  
Pilar Puig-Serra ◽  
Pablo Menendez ◽  
Raul Torres-Ruiz ◽  
Sandra Rodriguez-Perales
Keyword(s):  
Cancer Therapy ◽  
High Efficiency ◽  
Oncolytic Viruses ◽  
Model Organisms ◽  
Chemical Genetic ◽  
Social Burden ◽  
Culture Models ◽  
Therapeutic Tools ◽  
Cancer Immunotherapeutic ◽  
Clinical Potential

Cancer is the second leading cause of death globally and remains a major economic and social burden. Although our understanding of cancer at the molecular level continues to improve, more effort is needed to develop new therapeutic tools and approaches exploiting these advances. Because of its high efficiency and accuracy, the CRISPR-Cas9 genome editing technique has recently emerged as a potentially powerful tool in the arsenal of cancer therapy. Among its many applications, CRISPR-Cas9 has shown an unprecedented clinical potential to discover novel targets for cancer therapy and to dissect chemical-genetic interactions, providing insight into how tumours respond to drug treatment. Moreover, CRISPR-Cas9 can be employed to rapidly engineer immune cells and oncolytic viruses for cancer immunotherapeutic applications. Perhaps more importantly, the ability of CRISPR-Cas9 to accurately edit genes, not only in cell culture models and model organisms but also in humans, allows its use in therapeutic explorations. In this review, we discuss important considerations for the use of CRISPR/Cas9 in therapeutic settings and major challenges that will need to be addressed prior to its clinical translation for a complex and polygenic disease such as cancer.

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