scholarly journals Pooled CRISPR screening at high sensitivity with an empirically designed sgRNA library

2020 ◽  
Author(s):  
Luisa Henkel ◽  
Benedikt Rauscher ◽  
Barbara Schmitt ◽  
Jan Winter ◽  
Michael Boutros
Keyword(s):  
Experimental Design ◽  
Single Cell ◽  
Dynamic Range ◽  
High Sensitivity ◽  
Human Cells ◽  
Gene Essentiality ◽  
Cell Clones ◽  
Experimental Implementation ◽  
Genome Scale ◽  
Target Activity

ABSTRACTGiven their broad utility in functionally annotating genomes, the experimental design of genome-scale CRISPR screens can vary greatly and criteria for optimal experimental implementation and library composition are still emerging. In this study, we report advantages of conducting viability screens in selected Cas9 single cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg)RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a new genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes. In summary, we show how empirically designed libraries in combination with an optimised experimental design increase the dynamic range in gene essentiality screens at reduced library coverage.

scholarly journals Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Luisa Henkel ◽  
Benedikt Rauscher ◽  
Barbara Schmitt ◽  
Jan Winter ◽  
Michael Boutros
Keyword(s):  
Large Scale ◽  
Dynamic Range ◽  
High Sensitivity ◽  
Cell Types ◽  
Functional Dependencies ◽  
Protein Coding ◽  
Cell Clones ◽  
Experimental Implementation ◽  
Numerous Cell ◽  
Genome Scale

Abstract Background In recent years, large-scale genetic screens using the CRISPR/Cas9 system have emerged as scalable approaches able to interrogate gene function with unprecedented efficiency and specificity in various biological contexts. By this means, functional dependencies on both the protein-coding and noncoding genome of numerous cell types in different organisms have been interrogated. However, screening designs vary greatly and criteria for optimal experimental implementation and library composition are still emerging. Given their broad utility in functionally annotating genomes, the application and interpretation of genome-scale CRISPR screens would greatly benefit from consistent and optimal design criteria. Results We report advantages of conducting viability screens in selected Cas9 single-cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg) RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a novel genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes. Conclusion We show how empirically designed libraries in combination with an optimized experimental design increase the dynamic range in gene essentiality screens at reduced library coverage.

Slow-Scan CCD Observation of Backscattering Patterns in SEM

1992 ◽  
Vol 50 (2) ◽  
pp. 1308-1309
Author(s):  
F. Ouyang ◽  
D. A. Ray ◽  
O. L. Krivanek
Keyword(s):  
Electron Beam ◽  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Dynamic Range ◽  
High Sensitivity ◽  
Lens System ◽  
Wide Dynamic Range ◽  
Peltier Cooler ◽  
Diffraction Patterns ◽  
Scanning Electron

Electron backscattering Kikuchi diffraction patterns (BKDP) reveal useful information about the structure and orientation of crystals under study. With the well focused electron beam in a scanning electron microscope (SEM), one can use BKDP as a microanalysis tool. BKDPs have been recorded in SEMs using a phosphor screen coupled to an intensified TV camera through a lens system, and by photographic negatives. With the development of fiber-optically coupled slow scan CCD (SSC) cameras for electron beam imaging, one can take advantage of their high sensitivity and wide dynamic range for observing BKDP in SEM.We have used the Gatan 690 SSC camera to observe backscattering patterns in a JEOL JSM-840A SEM. The CCD sensor has an active area of 13.25 mm × 8.83 mm and 576 × 384 pixels. The camera head, which consists of a single crystal YAG scintillator fiber optically coupled to the CCD chip, is located inside the SEM specimen chamber. The whole camera head is cooled to about -30°C by a Peltier cooler, which permits long integration times (up to 100 seconds).

Development of imaging plate for a TEM and its characteristics

1989 ◽  
Vol 47 ◽  
pp. 100-101
Author(s):  
N. Mori ◽  
T. Oikawa ◽  
Y. Harada ◽  
J. Miyahara ◽  
T. Matsuo
Keyword(s):  
Dynamic Range ◽  
Protective Layer ◽  
High Sensitivity ◽  
Imaging Plate ◽  
Phosphor Layer ◽  
Imaging Device ◽  
X Ray Imaging ◽  
New Type ◽  
Basic Characteristics ◽  
Reading System

The Imaging Plate (IP) is a new type imaging device, which was developed for diagnostic x ray imaging. We have reported that usage of the IP for a TEM has many merits; those are high sensitivity, wide dynamic range, and good linearity. However in the previous report the reading system was prototype drum-type-scanner, and IP was also experimentally made, which phosphor layer was 50μm thick with no protective layer. So special care was needed to handle them, and they were used only to make sure the basic characteristics. In this article we report the result of newly developed reading, printing system and high resolution IP for practical use. We mainly discuss the characteristics of the IP here. (Precise performance concerned with the reader and other system are reported in the other article.)Fig.1 shows the schematic cross section of the IP. The IP consists of three parts; protective layer, phosphor layer and support.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

scholarly journals Differentiating and quantifying exosome secretion from a single cell using quasi-bound states in the continuum

Nanophotonics ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1081-1086 ◽  
Author(s):  
Abdoulaye Ndao ◽  
Liyi Hsu ◽  
Wei Cai ◽  
Jeongho Ha ◽  
Junhee Park ◽  
...  
Keyword(s):  
Single Cell ◽  
Optical Sensors ◽  
Figure Of Merit ◽  
Bound States ◽  
Single Cell Analysis ◽  
Optical Cavity ◽  
High Sensitivity ◽  
Cell Secretion ◽  
The Continuum ◽  
Refractive Index Detection

AbstractOne of the key challenges in biology is to understand how individual cells process information and respond to perturbations. However, most of the existing single-cell analysis methods can only provide a glimpse of cell properties at specific time points and are unable to provide cell secretion and protein analysis at single-cell resolution. To address the limits of existing methods and to accelerate discoveries from single-cell studies, we propose and experimentally demonstrate a new sensor based on bound states in the continuum to quantify exosome secretion from a single cell. Our optical sensors demonstrate high-sensitivity refractive index detection. Because of the strong overlap between the medium supporting the mode and the analytes, such an optical cavity has a figure of merit of 677 and sensitivity of 440 nm/RIU. Such results facilitate technological progress for highly conducive optical sensors for different biomedical applications.

scholarly journals High-frequency rectifiers based on type-II Dirac fermions

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Libo Zhang ◽  
Zhiqingzi Chen ◽  
Kaixuan Zhang ◽  
Lin Wang ◽  
Huang Xu ◽  
...  
Keyword(s):  
High Frequency ◽  
Dynamic Range ◽  
High Sensitivity ◽  
High Dynamic Range ◽  
Anisotropy Ratio ◽  
Dirac Fermions ◽  
Topological States ◽  
Topological Semimetals ◽  
Polarized Surface ◽  
Symmetry Protected

AbstractThe advent of topological semimetals enables the exploitation of symmetry-protected topological phenomena and quantized transport. Here, we present homogeneous rectifiers, converting high-frequency electromagnetic energy into direct current, based on low-energy Dirac fermions of topological semimetal-NiTe2, with state-of-the-art efficiency already in the first implementation. Explicitly, these devices display room-temperature photosensitivity as high as 251 mA W−1 at 0.3 THz in an unbiased mode, with a photocurrent anisotropy ratio of 22, originating from the interplay between the spin-polarized surface and bulk states. Device performances in terms of broadband operation, high dynamic range, as well as their high sensitivity, validate the immense potential and unique advantages associated to the control of nonequilibrium gapless topological states via built-in electric field, electromagnetic polarization and symmetry breaking in topological semimetals. These findings pave the way for the exploitation of topological phase of matter for high-frequency operations in polarization-sensitive sensing, communications and imaging.

High-sensitivity and wide-dynamic-range 10 Gbit∕s APD∕preamplifier optical receiver module

2002 ◽  
Vol 38 (13) ◽  
pp. 650 ◽  
Author(s):  
H. Matsuda ◽  
A. Miura ◽  
H. Irie ◽  
S. Tanaka ◽  
K. Ito ◽  
...  
Keyword(s):  
Dynamic Range ◽  
High Sensitivity ◽  
Optical Receiver ◽  
Wide Dynamic Range ◽  
Receiver Module
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