Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR-Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for ...the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by its comprehensive pipeline, which contains a series of functions for analyzing CRISPR screen data. This protocol explains how to use MAGeCKFlute to perform quality control (QC), normalization, batch effect removal, copy-number bias correction, gene hit identification and downstream functional enrichment analysis for CRISPR screens. We also describe gene identification and data analysis in CRISPR screens involving drug treatment. Completing the entire MAGeCKFlute pipeline requires ~3 h on a desktop computer running Linux or Mac OS with R support.
CRISPR-Cas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a ...single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPR-Cas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.
Palladium diselenide (PdSe2), a peculiar noble metal dichalcogenide, has emerged as a new two-dimensional material with high predicted carrier mobility and a widely tunable band gap for device ...applications. The inherent in-plane anisotropy endowed by the pentagonal structure further renders PdSe2 promising for novel electronic, photonic, and thermoelectric applications. However, the direct synthesis of few-layer PdSe2 is still challenging and rarely reported. Here, we demonstrate that few-layer, single-crystal PdSe2 flakes can be synthesized at a relatively low growth temperature (300 °C) on sapphire substrates using low-pressure chemical vapor deposition (CVD). The well-defined rectangular domain shape and precisely determined layer number of the CVD-grown PdSe2 enable us to investigate their layer-dependent and in-plane anisotropic properties. The experimentally determined layer-dependent band gap shrinkage combined with first-principle calculations suggest that the interlayer interaction is weaker in few-layer PdSe2 in comparison with that in bulk crystals. Field-effect transistors based on the CVD-grown PdSe2 also show performances comparable to those based on exfoliated samples. The low-temperature synthesis method reported here provides a feasible approach to fabricate high-quality few-layer PdSe2 for device applications.
All‐polymer solar cells (all‐PSCs) have received attention in recent years for their desirable properties in power conversion efficiency and long‐term operational stability. However, it is still a ...big challenge to acquire an “ideal” vertical‐phase distribution of polymer/polymer blends due to the non‐ideal molecular conformations and mixing behaviors. Herein, a ternary‐assisted sequential solution deposition (SSD) strategy is adopted to regulate the vertical compositional profile of all‐PSCs. A favorable acceptor(donor)‐enriched phase near the cathode(anode) can be obtained by a ternary‐assisted SSD strategy. With such a compositional profile, the exciton yield and carrier density can be enhanced by the vertical component gradient. Remarkably, the non‐geminate recombination is suppressed with an improved exciton diffusion length (15.36 nm) that delivers an outstanding power conversion efficiency over 16% of the ternary PM6/PY‐IT:PDI‐2T SSD devices. This work demonstrates the success of ternary‐assisted SSD strategy in reorganizing the vertical‐phase distribution, which provides a feasible route for a potential ternary device construction toward efficient all‐polymer photovoltaics.
A vertical compositional gradient within the active layer with a donor‐enriched active‐anode interface and an acceptor‐enriched active‐cathode interface can be achieved by sequential solution deposition treatment in PM6/PY‐IT:PDI‐2T device. As a result, charge transfer properties and exciton diffusion length are promoted with suppressed non‐geminate recombination to deliver an outstanding power conversion efficiency of 16% in the all‐polymer solar cells, which are verified with transient absorption, time‐resolved photoluminescence, and capacitance‐voltage measurements.
High-throughput CRISPR screens have shown great promise in functional genomics. We present MAGeCK-VISPR, a comprehensive quality control (QC), analysis, and visualization workflow for CRISPR screens. ...MAGeCK-VISPR defines a set of QC measures to assess the quality of an experiment, and includes a maximum-likelihood algorithm to call essential genes simultaneously under multiple conditions. The algorithm uses a generalized linear model to deconvolute different effects, and employs expectation-maximization to iteratively estimate sgRNA knockout efficiency and gene essentiality. MAGeCK-VISPR also includes VISPR, a framework for the interactive visualization and exploration of QC and analysis results. MAGeCK-VISPR is freely available at http://bitbucket.org/liulab/mageck-vispr .
The Majorana fermion, which is its own antiparticle and obeys non-Abelian statistics, plays a critical role in topological quantum computing. It can be realized as a bound state at zero energy, ...called a Majorana zero mode (MZM), in the vortex core of a topological superconductor, or at the ends of a nanowire when both superconductivity and strong spin orbital coupling are present. A MZM can be detected as a zero-bias conductance peak (ZBCP) in tunneling spectroscopy. However, in practice, clean and robust MZMs have not been realized in the vortices of a superconductor because of contamination from impurity states or other closely packed Caroli–de Gennes-Matricon (CdGM) states, which hampers further manipulations of MZMs. Here, using scanning tunneling spectroscopy, we show that a ZBCP well separated from the other discrete CdGM states exists ubiquitously in the cores of free vortices in the defect-free regions of(Li0.84Fe0.16)OHFeSe, which has a superconducting transition temperature of 42 K. Moreover, a Dirac-cone-type surface state is observed by angle-resolved photoemission spectroscopy, and its topological nature is confirmed by band calculations. The observed ZBCP can naturally be attributed to a MZM arising from the chiral topological surface state of a bulk superconductor. Thus,(Li0.84Fe0.16)OHFeSeprovides an ideal platform for studying MZMs and topological quantum computing.
Rechargeable aluminum‐ion batteries (AIBs) are regarded as promising candidates for post‐lithium energy storage systems (ESSs). For addressing the critical issues in the current liquid AIB systems, ...here a flexible solid‐state AIB is established using a gel‐polymer electrolyte for achieving robust electrode–electrolyte interfaces. Different from utilization of solid‐state systems for alleviating the safety issues and enhancing energy density in lithium‐ion batteries, employment of polymeric electrolytes mainly focuses on addressing the essential problems in the liquid AIBs, including unstable internal interfaces induced by mechanical deformation and production of gases as well as unfavorable separators. Particularly, such gel electrolyte enables the solid‐state AIBs to present an ultra‐fast charge capability within 10 s at current density of 600 mA g−1. Meanwhile, an impressive specific capacity ≈120 mA h g−1 is obtained at current density of 60 mA g−1, approaching the theoretical limit of graphite‐based AIBs. In addition to the well‐retained electrochemical performance below the ice point, the solid‐state AIBs also hold great stability and safety under various critical conditions. The results suggest that such new prototype of solid‐state AIBs with robust electrode–electrolyte interfaces promises a novel strategy for fabricating stable and safe flexible ESSs.
Flexible stable solid‐state Al‐ion batteries are constructed using the gel‐polymer electrolyte, Al anode, and graphite cathode. The novel polymeric electrolyte substantially promotes the overall energy storage performance via addressing the unstable internal interfaces induced by mechanical deformation and production of gases as well as unfavorable separators in the liquid systems, suggesting new strategies for achieving advanced flexible energy storage devices.
The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation ...(CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies.
Summary
NAC transcription factors play important roles in plant growth, development and stress responses. Previously, we identified multiple NAC genes in soybean (Glycine max). Here, we identify the ...roles of two genes, GmNAC11 and GmNAC20, in stress responses and other processes. The two genes were differentially induced by multiple abiotic stresses and plant hormones, and their transcripts were abundant in roots and cotyledons. Both genes encoded proteins that localized to the nucleus and bound to the core DNA sequence CGTG/A. In the protoplast assay system, GmNAC11 acts as a transcriptional activator, whereas GmNAC20 functions as a mild repressor; however, the C‐terminal end of GmANC20 has transcriptional activation activity. Over‐expression of GmNAC20 enhances salt and freezing tolerance in transgenic Arabidopsis plants; however, GmNAC11 over‐expression only improves salt tolerance. Over‐expression of GmNAC20 also promotes lateral root formation. GmNAC20 may regulate stress tolerance through activation of the DREB/CBF–COR pathway, and may control lateral root development by altering auxin signaling‐related genes. GmNAC11 probably regulates DREB1A and other stress‐related genes. The roles of the two GmNAC genes in stress tolerance were further analyzed in soybean transgenic hairy roots. These results provide a basis for genetic manipulation to improve the agronomic traits of important crops.
Semitransparent organic photovoltaics (ST‐OPVs) have great potential for use in renewable energy technologies. In bulk‐heterojunction (BHJ) ST‐OPVs, a compromise is necessary between the visible ...light transmittance (VLT) and the power conversion efficiency (PCE). A sequential deposition (SD) strategy that involves individually depositing a polymer donor layer (D) and a small‐molecule acceptor layer (A) as the active layer is presented; where molecular diffusion occurring at the interfacial region results in a pseudo p–i–n structure. PBDB‐T‐2F(D)/Y6(A) ST‐OPVs are fabricated with different active layer thicknesses—at 115 nm, the SD (D:A/75:40 nm) and BHJ devices (D:A/1:1.2 w) provide the champion PCE of 12.91% (VLT of 14.5%) and 12.77% (VLT of 13.4%), respectively; at 85 nm, the SD (D:A/45:40 nm) and BHJ devices (D:A/1:1.2 w) provide a PCE of 12.22% (VLT of 22.2%) and 11.23% (VLT of 16.6%), respectively. This trend indicates SD devices have larger PCE and VLT values than the BHJ devices at a given active layer thickness, and the enhancements of PCE and VLT values by the SD structures against the BHJ structures become more pronounced as the active layer thickness reduced. The SD strategy provides a new approach for achieving ST‐OPVs with both high efficiency and high transparency.
In typical semitransparent organic photovoltaics (ST‐OPVs) that incorporate bulk heterojunction (BHJ) active layers, a compromise is made between the visible light transmittance (VLT) and power conversion efficiency (PCE). A new strategy with a sequential‐deposition (SD) active layer involving pseudo p–i–n structures provides ST‐OPVs with simultaneously higher PCE and VLT than that of the BHJ devices at the same layer thickness.
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