Making complex things simpler: modern tools to edit the plant genome

There are several technologies for plant genome editing, of which the most simple and universal is CRISPR/Cas. Currently, this technology is widely used for gene knockout, deleting genome fragments and inserting exogenous sequences in the plant genome. For each of these applications, many different types of genetic tools have been developed that are used by various research groups to solve specific problems. The CRISPR/Cas technology for plant genome editing is at an early stage of optimization, which is reflected by the ongoing search for the most effective, simple and flexible techniques. As a result, experimental work has to be preceded by a rather long and laborious process of selecting a genetic tool that will be optimal for a specific experimental task. In our review we describe the main variants of the CRISPR/Cas technology used to edit a plant genome. We classify them in terms of experimental tasks solved, major components and technology performance. In the first half of the review a detailed description of two major components of CRISPR/Cas technology – nuclease and guide RNA – is given, the effect of structural features of these elements on editing efficiency is analyzed. Experimental data on the relationship between editing efficiency and nucleotide sequence of guide RNA are generalized. We also give the characteristic for different variants of nucleases used for plant genome editing and discuss their benefits for different experimental purposes. In the second half of the review various strategies for expression of CRISPR/Cas elements in plant cells, in particular, advantages and disadvantages of stable transformation and transient expression, are discussed. The effect of various regulatory elements of genes encoding nuclease and guide RNA on editing efficiency is described. Special emphasis is placed on the techniques of increasing targeted gene replacement efficiency

Development and Test of a Single-Aperture 11 T Nb3Sn Demonstrator Dipole for LHC Upgrades

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Quench Protection Study of a Single-Aperture 11 T Nb3Sn Demonstrator Dipole for LHC Upgrades

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Field Quality Measurements in a Single-Aperture 11 T Nb3Sn Demonstrator Dipole for LHC Upgrades

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Development of Rutherford-type cables for high field accelerator magnets at Fermilab

Fermilab's cabling facility has been upgraded to a maximum capability of 42 strands. This facility is being used to study the effect of cabling on the performance of the various strands, and for the development and fabrication of cables in support of the ongoing magnet R&D programs. Rutherford cables of various geometries, packing factors, with and without a stainless steel core, were fabricated out of Cu alloys, NbTi, Nb{sub 3}Al, and various Nb{sub 3}Sn strands. The parameters of the upgraded cabling machine and results of cable R&D efforts at Fermilab are reported

Development of Nb3Sn 11 T single aperture demonstrator dipole for LHC upgrades

The LHC collimation upgrade foresees additional collimators installed in dispersion suppressor regions. To obtain the necessary space for the collimators, a solution based on the substitution of LHC main dipoles for stronger dipoles is being considered. CERN and FNAL have started a joint program to demonstrate the feasibility of Nb{sub 3}Sn technology for this purpose. The goal of the first phase is the design and construction of a 2-m long single-aperture demonstrator magnet with a nominal field of 11 T at 11.85 kA with 20% margin. This paper describes the magnetic and mechanical design of the demonstrator magnet and summarizes its design parameters

Reconstruction of recombination sites in genomic structures of the strains of genotype 6 of hepatitis C virus

The encoded portion of the complete genomes of 46 strains of the genotype 6 of hepatitis C virus through bioinformatics RDP programs complex group of 6 recombinants strains was identified, in which 7 recombination sites were fixed. Strains correspond to the three-recombinant HCV subtypes: 6a, 6b and 61. For each of the identified recombinant we defined parent strains from which they can be obtained. Three recombinants were obtained from parent strains of the same subtype (homologous inside subgenotypic recombination). For the remaining three recombinants parent strains were members of three different subtypes (between subgenotypic recombination).In one strain we identified a unique recombination site in a highly conservative NS3 gene. Most of the recombination sites occurred in the region of the structural genes C, E1 and E2, and in the area of non-structural genes NS5a and NS5b.In the recombinant strain DQ480518-6a two recombination site were identified. One site is located in the structural and nonstructural genes (E2 + NS1 + NS2), and a second one in non-structural region. Dimensions of recombination sites can vary from 86 to 1072 nucleotide bases. The study identified "hot spots" of recombination in the strains of genotype 6 of hepatitis C virus. The recombinants were found in the population of the three countries: the United States (from the serum of an immigrant), Hong Kong and China

Bioinformational analysis of Yersinia pseudotuberculosis IP32953 CRISPR/cas system

The results of this study include Yersinia pseudotuberculosis CRISPR/Cas system structure analysis. CRISPR/Cas system is a specific adaptive protection against heterogeneous genetic elements. The object of research was the complete genome of Y. pseudotuberculosis IP32953 (NC_006155). CRISPR/Cas system screening was performed by program modelling methods MacSyFinder ver. 1.0.2. CRISPR loci screening and analyzing were carried out by program package: CRISPR Recognition tool (CRT), CR1SP1: a CRISPR Interactive database, CRISPRFinder, and PilerCR. Spacer sequences were used in order to find protospacers in ACLAME, GenBank-Phage and RefSeq-Plasmid databases by BLASTn search algorithm. Protospacer sequences could be found in genomes of phages, plasmids and bacteria. In last case complete genomes of bacteria were analyzed by online-tool PHAST: PHAge Search Tool. Y. pseudotuberculosis IP329353 has CRISPR/Cas system that consists of one sequence of cas-genes and three loci. These loci are far away from each other. Locus YP1 is situated in close proximity to cas-genes. Protospacers were found in genomes of Y. pseudotuberculosis PB1/+, Y. intermedia Y228, Y. similis str. 228, Salmonella phage, Enterobacteria phage, Y. pseudotuberculosis 1P32953 plasmid pYV and plasmid of Y. pseudotuberculosis 1P31758. Thus, the combination of four program methods allows finding CRISPR/Cas system more precisely. Spacer sequences could be used for protospacer screening

Design and Fabrication of a Single-Aperture 11T Nb3Sn Dipole Model for LHC Upgrades

The planned upgrade of the LHC collimation system includes additional collimators to be installed in the dispersion suppressor areas of points 2, 3 and 7. To provide the necessary longitudinal space for the collimators, a replacement of 8.33 T Nb-Ti LHC main dipoles with 11 T dipoles based on Nb{sub 3}Sn superconductor compatible with the LHC lattice and main systems is being considered. To demonstrate this possibility FNAL and CERN have started a joint program to develop a 2 m long single-aperture dipole magnet with the nominal field of 11 T at {approx}11.85 kA current and 60 mm bore. This paper describes the demonstrator magnet magnetic and mechanical designs and analysis, coil fabrication procedure. The Nb{sub 3}Sn strand and cable parameters and test results are also reported

Testing of Nb3Sn quadrupole coils using magnetic mirror structure

This paper describes the design and parameters of a quadrupole mirror structure for testing the mechanical, thermal and quench performance of single shell-type superconducting quadrupole coils at field, current and force levels similar to that of real magnet. The concept was experimentally verified by testing two quadrupole coils, previously used in quadrupole models, in the developed mirror structure in the temperature range from 4.5 to 1.9 K. The coils were instrumented with voltage taps, heaters, and strain gauges to monitor their mechanical and thermal properties and quench performance. A new quadrupole coil made of improved Nb{sub 3}Sn RRP-108/127 strand and cable insulation based on E-glass tape was also tested using this structure. The fabrication and test results of the quadrupole mirror models are reported and discussed