High-Performance Allosteric Conditional Guide RNAs for Mammalian Cell-Selective Regulation of CRISPR/Cas

2021 ◽  
Author(s):  
Lisa M. Hochrein ◽  
Heyun Li ◽  
Niles A. Pierce
Keyword(s):  
Mammalian Cell ◽  
High Performance ◽  
Guide Rnas

scholarly journals CRISPR-SE: a brute force search engine for CRISPR design

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Bin Li ◽  
Poshen B Chen ◽  
Yarui Diao
Keyword(s):  
Search Engine ◽  
Sequence Alignment ◽  
Search Engines ◽  
High Performance ◽  
Design Tools ◽  
Brute Force ◽  
Guide Rnas ◽  
Speed Up ◽  
Brute Force Approach

Abstract CRISPR is a revolutionary genome-editing tool that has been broadly used and integrated within novel biotechnologies. A major component of existing CRISPR design tools is the search engines that find the off-targets up to a predefined number of mismatches. Many CRISPR design tools adapted sequence alignment tools as the search engines to speed up the process. These commonly used alignment tools include BLAST, BLAT, Bowtie, Bowtie2 and BWA. Alignment tools use heuristic algorithm to align large amount of sequences with high performance. However, due to the seed-and-extend algorithms implemented in the sequence alignment tools, these methods are likely to provide incomplete off-targets information for ultra-short sequences, such as 20-bp guide RNAs (gRNA). An incomplete list of off-targets sites may lead to erroneous CRISPR design. To address this problem, we derived four sets of gRNAs to evaluate the accuracy of existing search engines; further, we introduce a search engine, namely CRISPR-SE. CRISPR-SE is an accurate and fast search engine using a brute force approach. In CRISPR-SE, all gRNAs are virtually compared with query gRNA, therefore, the accuracies are guaranteed. We performed the accuracy benchmark with multiple search engines. The results show that as expected, alignment tools reported an incomplete and varied list of off-target sites. CRISPR-SE performs well in both accuracy and speed. CRISPR-SE will improve the quality of CRISPR design as an accurate high-performance search engine.

Rapid Development of High-Performance Stable Mammalian Cell Lines for Improved Clinical Development

2009 ◽  
Vol 7 (4) ◽  
pp. 30-36 ◽  
Author(s):  
Jose Varghese ◽  
Washington Alves ◽  
Benjamin Brill ◽  
Matt Wallace ◽  
David Calabrese ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
High Performance ◽  
Rapid Development ◽  
Clinical Development ◽  
Mammalian Cell Lines

scholarly journals Functional dissection of HGGT and HPT in barley vitamin E biosynthesis via CRISPR/Cas9-enabled genome editing

2020 ◽  
Vol 126 (5) ◽  
pp. 929-942 ◽  
Author(s):  
Zhanghui Zeng ◽  
Ning Han ◽  
Cuicui Liu ◽  
B Buerte ◽  
Chenlu Zhou ◽  
...  
Keyword(s):  
Vitamin E ◽  
Genome Editing ◽  
High Performance ◽  
Gene Knockout ◽  
Starch Content ◽  
Guide Rnas ◽  
Targeted Mutation ◽  
Barley Grains ◽  
Transient Overexpression ◽  
High Level

Abstract Background and Aims Vitamin E (tocochromanol) is a lipid-soluble antioxidant and an essential nutrient for human health. Among cereal crops, barley (Hordeum vulgare) contains a high level of vitamin E, which includes both tocopherols and tocotrienols. Although the vitamin E biosynthetic pathway has been characterized in dicots, such as Arabidopsis, which only accumulate tocopherols, knowledge regarding vitamin E biosynthesis in monocots is limited because of the lack of functional mutants. This study aimed to obtain gene knockout mutants to elucidate the genetic control of vitamin E composition in barley. Methods Targeted knockout mutations of HvHPT and HvHGGT in barley were created with CRISPR/Cas9-enabled genome editing. High-performance liquid chromatography (HPLC) was performed to analyse the content of tocochromanol isomers in transgene-free homozygous Hvhpt and Hvhggt mutants. Key Results Mutagenesis efficiency among T0 regenerated plantlets was 50–65 % as a result of two simultaneously expressed guide RNAs targeting each gene; most of the mutations were stably inherited by the next generation. The transgene-free homozygous mutants of Hvhpt and Hvhggt exhibited decreased grain size and weight, and the HvHGGT mutation led to a shrunken phenotype and significantly lower total starch content in grains. HPLC analysis revealed that targeted mutation of HvHPT significantly reduced the content of both tocopherols and tocotrienols, whereas mutations in HvHGGT completely blocked tocotrienol biosynthesis in barley grains. Transient overexpression of an HvHPT homologue in tobacco leaves significantly increased the production of γ- and δ-tocopherols, which may partly explain why targeted mutation of HvHPT in barley grains did not eliminate tocopherol production. Conclusions Our results functionally validated that HvHGGT is the only committed gene for the production of tocotrienols, whereas HvHPT is partly responsible for tocopherol biosynthesis in barley.

A High Performance Energy Analyzer for Use in Electron Scanning Microscopy

1969 ◽  
Vol 27 ◽  
pp. 14-15
Author(s):  
A. V. Crewe ◽  
M. Isaacson ◽  
D. Johnson
Keyword(s):  
High Performance ◽  
Vertical Plane ◽  
Median Plane ◽  
Electron Gun ◽  
Uniform Field ◽  
Loss Mechanism ◽  
Electron Scanning Microscopy ◽  
Sector Type ◽  
Double Focusing ◽  
Electron Energy Loss

A double focusing magnetic spectrometer has been constructed for use with a field emission electron gun scanning microscope in order to study the electron energy loss mechanism in thin specimens. It is of the uniform field sector type with curved pole pieces. The shape of the pole pieces is determined by requiring that all particles be focused to a point at the image slit (point 1). The resultant shape gives perfect focusing in the median plane (Fig. 1) and first order focusing in the vertical plane (Fig. 2).

Vacuum System to Minimize the Specimen Contamination of High-Performance EM

1977 ◽  
Vol 35 ◽  
pp. 68-69
Author(s):  
N. Yoshimura ◽  
K. Shirota ◽  
T. Etoh
Keyword(s):  
Electron Microscope ◽  
High Speed ◽  
High Performance ◽  
High Vacuum ◽  
Vacuum System ◽  
Pump System ◽  
Pumping System ◽  
Diffusion Pump ◽  
Almost All ◽  
Cascade Type

One of the most important requirements for a high-performance EM, especially an analytical EM using a fine beam probe, is to prevent specimen contamination by providing a clean high vacuum in the vicinity of the specimen. However, in almost all commercial EMs, the pressure in the vicinity of the specimen under observation is usually more than ten times higher than the pressure measured at the punping line. The EM column inevitably requires the use of greased Viton O-rings for fine movement, and specimens and films need to be exchanged frequently and several attachments may also be exchanged. For these reasons, a high speed pumping system, as well as a clean vacuum system, is now required. A newly developed electron microscope, the JEM-100CX features clean high vacuum in the vicinity of the specimen, realized by the use of a CASCADE type diffusion pump system which has been essentially improved over its predeces- sorD employed on the JEM-100C.

Use of the Magnetostatic Analogue In Electromagnetic Lens Engineering

1970 ◽  
Vol 28 ◽  
pp. 360-361
Author(s):  
John W. Coleman
Keyword(s):  
Boundary Conditions ◽  
High Performance ◽  
Force Fields ◽  
Optical Design ◽  
Direct Conversion ◽  
Design Engineering ◽  
Design Data ◽  
Scalar Potentials ◽  
Geometric Elements ◽  
At Will

In the design engineering of high performance electromagnetic lenses, the direct conversion of electron optical design data into drawings for reliable hardware is oftentimes difficult, especially in terms of how to mount parts to each other, how to tolerance dimensions, and how to specify finishes. An answer to this is in the use of magnetostatic analytics, corresponding to boundary conditions for the optical design. With such models, the magnetostatic force on a test pole along the axis may be examined, and in this way one may obtain priority listings for holding dimensions, relieving stresses, etc..The development of magnetostatic models most easily proceeds from the derivation of scalar potentials of separate geometric elements. These potentials can then be conbined at will because of the superposition characteristic of conservative force fields.

Prospects for convergent beam electron diffraction with a 200kV cold field-emission transmission electron microscope

1991 ◽  
Vol 49 ◽  
pp. 466-467
Author(s):  
J W Steeds ◽  
R Vincent
Keyword(s):  
Field Emission ◽  
High Performance ◽  
Small Diameter ◽  
Convergent Beam Electron Diffraction ◽  
Zone Axis ◽  
Medium Voltage ◽  
Transmission Electron ◽  
Good Crystallinity ◽  
Special Modification ◽  
Convergent Beam

We review the analytical powers which will become more widely available as medium voltage (200-300kV) TEMs with facilities for CBED on a nanometre scale come onto the market. Of course, high performance cold field emission STEMs have now been in operation for about twenty years, but it is only in relatively few laboratories that special modification has permitted the performance of CBED experiments. Most notable amongst these pioneering projects is the work in Arizona by Cowley and Spence and, more recently, that in Cambridge by Rodenburg and McMullan.There are a large number of potential advantages of a high intensity, small diameter, focussed probe. We discuss first the advantages for probes larger than the projected unit cell of the crystal under investigation. In this situation we are able to perform CBED on local regions of good crystallinity. Zone axis patterns often contain information which is very sensitive to thickness changes as small as 5nm. In conventional CBED, with a lOnm source, it is very likely that the information will be degraded by thickness averaging within the illuminated area.

Current State of Biological High-Resolution Scanning Electron Microscopy

1988 ◽  
Vol 46 ◽  
pp. 180-181 ◽  
Author(s):  
Klaus-Ruediger Peters
Keyword(s):  
High Performance ◽  
Low Voltage ◽  
Backscattered Electrons ◽  
Lens Position ◽  
Low Voltage Operation ◽  
Signal Collection ◽  
Probe Size ◽  
Electron Microscopes ◽  
Scanning Electron Microscopes ◽  
Scanning Electron

A new generation of high performance field emission scanning electron microscopes (FSEM) is now commercially available (JEOL 890, Hitachi S 900, ISI OS 130-F) characterized by an "in lens" position of the specimen where probe diameters are reduced and signal collection improved. Additionally, low voltage operation is extended to 1 kV. Compared to the first generation of FSEM (JE0L JSM 30, Hitachi S 800), which utilized a specimen position below the final lens, specimen size had to be reduced but useful magnification could be impressively increased in both low (1-4 kV) and high (5-40 kV) voltage operation, i.e. from 50,000 to 200,000 and 250,000 to 1,000,000 x respectively.At high accelerating voltage and magnification, contrasts on biological specimens are well characterized1 and are produced by the entering probe electrons in the outmost surface layer within -vl nm depth. Backscattered electrons produce only a background signal. Under these conditions (FIG. 1) image quality is similar to conventional TEM (FIG. 2) and only limited at magnifications >1,000,000 x by probe size (0.5 nm) or non-localization effects (%0.5 nm).

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