scholarly journals The MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and Overexpression CRISPR-Cas9 Toolkit

2021 ◽  
Author(s):  
Bjorn DM Bean ◽  
Malcolm Whiteway ◽  
Vincent JJ Martin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Efficiency ◽  
Model Organism ◽  
Basic Research ◽  
Expression Vectors ◽  
Selectable Markers ◽  
Guide Rna ◽  
One Step ◽  
Selection For ◽  
The One

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful CRISPR-Cas9-based genome editing tools we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit with three components: (i) a set of optimized S. pyogenes Cas9-guide RNA (gRNA) expression vectors with five selectable markers and the option to either pre-clone or co-transform the gRNAs; (ii) vectors for the one-step construction of integration cassettes expressing an untagged or GFP/RFP/HA-tagged gene of interest at one of three levels, supporting localization and overexpression studies; and (iii) integration cassettes containing moderately expressed GFP- or RFP-tagged compartmental markers for colocalization experiments. These components allow rapid, high efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle to simplify yeast research

Research on Rubber One Step Injection Molding Technology and Equipment

2012 ◽  
Vol 501 ◽  
pp. 344-348
Author(s):  
Yi Zhou Pei ◽  
Bai Yuan Lv
Keyword(s):  
Injection Molding ◽  
High Efficiency ◽  
Low Cost ◽  
New Technology ◽  
Laboratory Research ◽  
Molding Method ◽  
Good Product ◽  
One Step ◽  
The One ◽  
Good Product Quality

Through the laboratory research on the one step injection molding technology, as well as a series of industrialization researches on the injection tire capsule, injection oilfield used screw drill rubber stator and injection solid tire, this thesis presents the principle of one step injection molding technology and proves the feasibility, practicability of this technology. Substituting the traditional compression molding method, one step injection molding is a new technology with high efficiency, good product quality and low cost; it also has a broad prospect of application and huge developing potential.

scholarly journals One-Step Construction of Multi-Walled CNTs Loaded with Alpha-Fe2O3 Nanoparticles for Efficient Photocatalytic Properties

Materials ◽  
2021 ◽  
Vol 14 (11) ◽  
pp. 2820
Author(s):  
Jianle Xu ◽  
Qiang Wen ◽  
Xiao Zhang ◽  
Yinhui Li ◽  
Zeyue Cui ◽  
...  
Keyword(s):  
Visible Light ◽  
High Efficiency ◽  
Separation Efficiency ◽  
Dye Degradation ◽  
Step Method ◽  
Visible Light Photocatalyst ◽  
Defect Sites ◽  
Multi Walled Carbon Nanotubes ◽  
One Step ◽  
The One

The aggregation and the rapid restructuring of the photoinduced electron−hole pairs restructuring in the process of photoelectric response remains a great challenge. In this study, a kind of Multi-walled carbon nanotubes loaded Alpha-Fe2O3 (CNTs/α-Fe2O3) heterostructure composite is successfully prepared via the one-step method. Due to the synergistic effect in the as-prepared CNTs/α-Fe2O3, the defect sites and oxygen-containing functional groups of CNTs can dramatically improve the interface charge separation efficiency and prevent the aggregation of α-Fe2O3. The improved photocurrent and enhanced hole–electron separation rate in the CNTs/α-Fe2O3 is obtained, and the narrower band gap is measured to be 2.8 ev with intensive visible-light absorption performance. Thus, the CNTs/α-Fe2O3 composite serves as an excellent visible light photocatalyst and exhibits an outstanding photocatalytic activity for the cationic dye degradation of rhodamine B (RhB). This research supplies a fresh application area forα-Fe2O3 photocatalyst and initiates a new approach for design of high efficiency photocatalytic materials.

scholarly journals CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Mengwan Li ◽  
Xuye Lang ◽  
Marcos Moran Cabrera ◽  
Sawyer De Keyser ◽  
Xiyan Sun ◽  
...  
Keyword(s):  
Kluyveromyces Marxianus ◽  
Shikimate Pathway ◽  
Pathway Engineering ◽  
Fuel Additive ◽  
Range Analysis ◽  
Production Route ◽  
Fragrance Compound ◽  
One Step ◽  
Selection For ◽  
The One

Abstract Background 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. Results We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 33-factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7G141S, results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. Conclusions The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

scholarly journals Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1121
Author(s):  
Marco Incarbone ◽  
Hélene Scheer ◽  
Jean-Michel Hily ◽  
Lauriane Kuhn ◽  
Mathieu Erhardt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Arabidopsis Thaliana ◽  
High Efficiency ◽  
Wide Spectrum ◽  
Model Organism ◽  
Plant Viruses ◽  
Tomato Bushy Stunt Virus ◽  
Defensive Reaction ◽  
Knock Out ◽  
Replication Sites

Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant–microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant–virus interactions in complement to Saccharomyces cerevisiae.

Sulfuryl Fluoride Promoted Thiocyanation of Alcohols: A Practical Method for Preparing Thiocyanates

Synlett ◽  
2020 ◽  
Vol 31 (14) ◽  
pp. 1413-1417
Author(s):  
Chengrong Ding ◽  
Guofu Zhang ◽  
Lidi Xuan ◽  
Yiyong Zhao
Keyword(s):  
Ethyl Acetate ◽  
Functional Group ◽  
High Efficiency ◽  
Bond Cleavage ◽  
Ammonium Thiocyanate ◽  
Practical Method ◽  
Sulfuryl Fluoride ◽  
Mild Conditions ◽  
One Step ◽  
The One

A novel SO2F2-promoted thiocyanation method for the one-step synthesis of thiocyanates through C–O bond cleavage of readily available alcohols with ammonium thiocyanate as the thiocyanating agent was developed. The method avoids the use of additional catalyst, and a variety of (hetero)arene, alkene and aliphatic alcohols reacted with high efficiency in ethyl acetate under mild conditions to afford the corresponding thiocyanates in excellent to quantitative yields with broad functional-group compatibility.

scholarly journals Saccharomyces cerevisiae-Based Molecular Tool Kit for Manipulation of Genes from Gram-Negative Bacteria

2006 ◽  
Vol 72 (7) ◽  
pp. 5027-5036 ◽  
Author(s):  
Robert M. Q. Shanks ◽  
Nicky C. Caiazza ◽  
Shannon M. Hinsa ◽  
Christine M. Toutain ◽  
George A. O'Toole
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Efficiency ◽  
Opportunistic Pathogen ◽  
Single Step ◽  
Expression Vectors ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Recombination Proteins ◽  
Wide Range

ABSTRACT A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.

scholarly journals CRISPR RNA-guided integrases for high-efficiency and multiplexed bacterial genome engineering

2020 ◽  
Author(s):  
Phuc Leo H. Vo ◽  
Carlotta Ronda ◽  
Sanne E. Klompe ◽  
Ethan E. Chen ◽  
Christopher Acree ◽  
...  
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Computational Algorithm ◽  
Bacterial Genome ◽  
Expression Vectors ◽  
Type I ◽  
Dna Integration ◽  
Guide Rna ◽  
Marker Free ◽  
Rna Design

Tn7-like transposons are pervasive mobile genetic elements in bacteria that mobilize using heteromeric transposase complexes comprising distinct targeting modules. We recently described a Tn7-like transposon from Vibrio cholerae that employs a Type I-F CRISPR–Cas system for RNA-guided transposition, in which Cascade directly recruits transposition proteins to integrate donor DNA downstream of genomic target sites complementary to CRISPR RNA. However, the requirement for multiple expression vectors and low overall integration efficiencies, particularly for large genetic payloads, hindered the practical utility of the transposon. Here, we present a significantly improved INTEGRATE (insertion of transposable elements by guide RNA-assisted targeting) system for targeted, multiplexed, and marker-free DNA integration of up to 10 kilobases at ~100% efficiency. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplex insertions in three genomic loci, and facile multi-loci deletions when combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in other biomedically- and industrially-relevant bacteria, and developed an accessible computational algorithm for guide RNA design. This work establishes INTEGRATE as a versatile and portable tool that enables multiplex and kilobase-scale genome engineering.

scholarly journals Fast, Efficient, and Precise Gene Editing in the MossPhyscomitrella patens

2019 ◽  
Author(s):  
Peishan Yi ◽  
Gohta Goshima
Keyword(s):  
Gene Editing ◽  
High Efficiency ◽  
Physcomitrella Patens ◽  
Gene Knockout ◽  
Model Organism ◽  
Plant Evolution ◽  
High Rate ◽  
Ideal System ◽  
Redundant Genes ◽  
One Step

AbstractRecent years, the bryophyte mossPhyscomitrella patenshas become an emerging model organism for studying conserved signaling pathways and developmental processes during plant evolution. Its short life cycle, ease of cultivation, and high rate of homologous recombination have made it an ideal system for genetic analysis. However, the presence of highly redundant genes and the difficulty of isolating hypomorphic mutants have limited its broader use. Here we developed a simple, fast, and efficient method to generate customized mutants inP. patens.We show that transient cotransformation of CRISPR/Cas9 and oligonucleotide templates enables microindel knock-in with high efficiency and accuracy. Using this method, we generated strains carrying various types of mutations, including amino acid substitution, out-of-frame deletion/insertion, splice site alteration, and small tag integration. We also demonstrate that multiplex gene editing can be efficiently achieved to generate putative null and hypomorphic mutants of redundant genes in one step. Thus our method will not only simplify multiple-gene knockout, but also allows the generation of hypomorphic mutants of genes of interest, especially those that are essential for viability.

scholarly journals Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

2015 ◽  
Vol 112 (11) ◽  
pp. 3570-3575 ◽  
Author(s):  
Kabin Xie ◽  
Bastian Minkenberg ◽  
Yinong Yang
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Crop Improvement ◽  
Processing System ◽  
Basic Research ◽  
Molecular Therapy ◽  
General Strategy ◽  
Trna Processing ◽  
Guide Rna

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.

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