Knockout Events Research Articles

Reconstitution of the Early Stage of Chetomin Biosynthesis in Aspergillus fumigatus Leads to the Production of Epipolythiodioxopiperazines.

Using CRISPR-Cas9 technology and a microhomology-mediated end-joining repair system, we substituted genes of the gliotoxin pathway in Aspergillus fumigatus with genes responsible for chetomin biosynthesis from Chaetomium cochliodes, leading to the production of three new epipolythiodioxopiperazines (ETPs). This work represents the first successful endeavor to produce ETPs in a non-native host. Additionally, the simultaneous disruption of five genes in a single transformation marks the most extensive gene knockout event in filamentous fungi to date.

Atomic displacement threshold energies and defect generation in GaN, AlN, and AlGaN: A high-throughput molecular dynamics investigation

Gallium nitride, aluminum nitride, and their ternary alloys form an important class of wide-bandgap semiconductors employed in a variety of applications, including radiation-hard electronics. To better understand the effects of irradiation in these materials, molecular dynamics simulations were employed to determine the threshold recoil energies to permanently displace atoms from crystalline sites. Threshold displacement energies were calculated with the lattices at 0 K. Thermal effects are found to lower the threshold energies by ∼1 eV. The threshold energy knockout events observed result in Frenkel pair defects. The electronic structure and dynamics of these Frenkel pair defects are analyzed and the consequences for device operation are discussed.

CRISPR-Cas9-induced targeted mutagenesis of feruloyl CoA 6′-hydroxylase gene reduces postharvest physiological deterioration in cassava roots

Cassava is a starch-rich, multipurpose root crop grown by millions of smallholder farmers in tropical and subtropical countries. Cassava cultivation is seriously constrained by post-harvest physiological deterioration (PPD) of roots that occurs within 24–72 h after harvest, making them unpalatable and unmarketable. The inevitable wounding of roots during harvest triggers an oxidative burst throughout the root together with the accumulation of secondary metabolites with scopoletin being the most abundant. Blue-black discoloration and vascular streaking observed during PPD occur due to over-accumulation of scopoletin whose biosynthesis involves key enzymes including feruloyl CoA 6′-hydroxylase.We applied the CRISPR-Cas9 system for targeted mutagenesis of feruloyl CoA 6′-hydroxylase genes (MeF6'H1, MeF6'H12 MeF6'H2 and MeF6'H3) in cassava to delay PPD. To generate cassava plants with enhanced root shelf-life, CRISPR-Cas9 cassettes with Cas9 gene and sgRNA targeting three genes; MeF6'H1, MeF6'H2, and MeF6'H3 were introduced into cassava via Agrobacterium-mediated transformation. Integration of transgenes in putative knockout plants were confirmed by PCR while targeted sanger sequencing revealed CRISPR-Cas9 induced mutations with insertion, deletions and substitution being reported upstream and downstream of the PAM sequence of target genes. Targeted for edition of dual genes exhibited significant reduction of scopoletin below HPLC detectable levels and had improved shelf-life compared to one gene knockout events and wild-type plants. Evaluation of phenotypic variations between mutant and wild-type cassava plants revealed mutation-event associated traits including stem and petiole depigmentation, auxiliary budding, claw-like leaf appearance, loss of apical dominance and leaf chlorosis. This study demonstrated an effective and feasible approach to extend cassava root shelf-life to stabilize yield production, prevent postharvest wastage, and improve farmers’ income.

The return barrier and return timer option with pricing under Lévy processes

This work introduces two new financial derivatives into the finance literature. The first is the return barrier option, which has emerged recently as a popular contract in the OTC markets. This contract is similar to a barrier option, but the knock-out event depends on an asset’s returns, rather than its level. Its direct exposure to jump risk offers protection from a key source of market incompleteness, with great potential value to hedgers and investors. The second contract is the return timer option, which is proposed in this work for its natural relation to the return barrier option, and by analogy to Timer Options written on realized volatility. This contract offers similar upside potential to a European option, but can be purchased at a substantial discount, a trade-off for bearing jump risk. Due to the dependence of these contracts on single (daily) return events, capturing jump risk is essential to their pricing, as diffusion-based models are unable to generate sufficiently heavy tails at short time horizons. Thus we provide analytical pricing formulas for the return barrier option, along with efficient and accurate numerical methods under Exponential Lévy dynamics, to capture the heavy-tailed returns. We then prove that the return timer option can be conveniently priced as a linear combination of return barriers, providing a consistent pricing framework for the combined family of derivatives. Given the recent trend in return barrier options in OTC markets, as well as the interesting mathematics required to price them, this work offers both practical and theoretical contributions to the literature.

Application of a novel strong promoter from Chinese fir (Cunninghamia lanceolate) in the CRISPR/Cas mediated genome editing of its protoplasts and transgenesis of rice and poplar.

Novel constitutive promoters are essential for plant biotechnology. Although in angiosperms, a number of promoters were applied in monocots or dicots genetic engineering, only a few promoters were used in gymnosperm. Here we identified two strong promoters (Cula11 and Cula08) from Chinese fir (C. lanceolate) by screening the transcriptomic data and preliminary promoter activity assays in tobacco. By using the newly established Chinese fir protoplast transient expression technology that enables in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII promoters were introduced into Chinese fir protoplasts, and CRISPR/Cas mediated gene knock-out event was successfully achieved. More importantly, compared with the commonly used promoters in the genetic engineering in angiosperms, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi promoter in transgenic rice, respectively, indicating its potential application in poplar and rice genetic engineering. Overall, the novel putative constitutive gene promoters reported here will have great potential application in gymnosperm and angiosperm biotechnology, and the transient gene expression system established here will serve as a useful tool for the molecular and genetic analyses of Chinese fir genes.

Atomic-displacement threshold energies and defect generation in irradiated β-Ga2O3: A first-principles investigation

Gallium oxide is an emerging wide-bandgap semiconductor with promise for applications in space systems that may be exposed to energetic particles. We use molecular dynamics simulations, based on first principles density-functional methods, to determine the nature and stability of the defects generated by atoms knocked-out by particle irradiation at near threshold energies (found to be 28±1eV for Ga and 14±1eV for O). For Ga atoms, several types of low energy knock-out events result in defect complexes, but the final structures depend critically on the initial displacement direction. In contrast, a vacancy plus a peroxide linkage occurs in all types of low energy knock-out events of O atoms. Based on energy-barrier calculations, there is a low (high) probability for Ga (O) defect recombination. The electronic structure of residual, relaxed defects generated by Ga knock-outs reveals defect levels near the band edges.

CRISPR/Cas9-Mediated Mutagenesis of the Granule-Bound Starch Synthase Gene in the Potato Variety Yukon Gold to Obtain Amylose-Free Starch in Tubers.

Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat. Its tubers are a rich source of dietary carbohydrates in the form of starch, which has many industrial applications. Starch is composed of two polysaccharides, amylose and amylopectin, and their ratios determine different properties and functionalities. Potato varieties with higher amylopectin have many food processing and industrial applications. Using Agrobacterium-mediated transformation, we delivered Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) reagents to potato (variety Yukon Gold) cells to disrupt the granule-bound starch synthase (gbssI) gene with the aim of eliminating the amylose component of starch. Lugol-Iodine staining of the tubers showed a reduction or complete elimination of amylose in some of the edited events. These results were further confirmed by the perchloric acid and enzymatic methods. One event (T2-7) showed mutations in all four gbss alleles and total elimination of amylose from the tubers. Viscosity profiles of the tuber starch from six different knockout events were determined using a Rapid Visco Analyzer (RVA), and the values reflected the amylopectin/amylose ratio. Follow-up studies will focus on eliminating the CRISPR components from the events and on evaluating the potential of clones with various amylose/amylopectin ratios for food processing and other industrial applications.

Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) as a Novel Regulator of Early Astroglial Differentiation.

Astrocytes are the most abundant cell type within the central nervous system (CNS) with various functions. Furthermore, astrocytes show a regional and developmental heterogeneity traceable with specific markers. In this study, the influence of the low-density lipoprotein receptor-related protein 1 (LRP1) on astrocytic maturation within the hippocampus was analyzed during development. Previous studies mostly focused on the involvement of LRP1 in the neuronal compartment, where the deletion caused hyperactivity and motor dysfunctions in knockout animals. However, the influence of LRP1 on glia cells is less intensively investigated. Therefore, we used a newly generated mouse model, where LRP1 is specifically deleted from GLAST-positive astrocytes co-localized with the expression of the reporter tdTomato to visualize recombination and knockout events in vivo. The influence of LRP1 on the maturation of hippocampal astrocytes was assessed with immunohistochemical stainings against stage-specific markers as well as on mRNA level with RT-PCR analysis. The examination revealed that the knockout induction caused a significantly decreased number of mature astrocytes at an early developmental timepoint compared to control animals. Additionally, the delayed maturation of astrocytes also caused a reduced activity of neurons within the hippocampus. As previous studies showed that the glial specification and maturation of astrocytes is dependent on the signaling cascades Ras/Raf/MEK/Erk and PI3K/Akt, the phosphorylation of the signaling molecules Erk1/2 and Akt was analyzed. The hippocampal tissue of LRP1-deficient animals at P21 showed a significantly decreased amount of activated Erk in comparison to control tissue leading to the conclusion that the activation of this signaling cascade is dependent on LRP1 in astrocytes, which in turn is necessary for proper maturation of astrocytes. Our results showed that the deletion of LRP1 at an early developmental timepoint caused a delayed maturation of astrocytes in the hippocampus based on an altered activation of the Ras/Raf/MEK/Erk signaling pathway. However, with ongoing development these effects were compensated and the number of mature astrocytes was comparable as well as the activity of neurons. Therefore, LRP1 acts as an early regulator of the differentiation and maturation of astrocytes within the hippocampus.

Deletion of LRP1 From Astrocytes Modifies Neuronal Network Activity in an in vitro Model of the Tripartite Synapse.

The low-density lipoprotein receptor-related protein 1 (LRP1) is a transmembrane receptor that binds over 40 potential ligands and is involved in processes such as cell differentiation, proliferation, and survival. LRP1 is ubiquitously expressed in the organism and enriched among others in blood vessels, liver, and the central nervous system (CNS). There, it is strongly expressed by neurons, microglia, immature oligodendrocytes, and astrocytes. The constitutive LRP1 knockout leads to embryonic lethality. Therefore, previous studies focused on conditional LRP1-knockout strategies and revealed that the deletion of LRP1 causes an increased differentiation of neural stem and precursor cells into astrocytes. Furthermore, astrocytic LRP1 is necessary for the degradation of Aβ and the reduced accumulation of amyloid plaques in Alzheimer’s disease. Although the role of LRP1 in neurons has intensely been investigated, the function of LRP1 with regard to the differentiation and maturation of astrocytes and their functionality is still unknown. To address this question, we generated an inducible conditional transgenic mouse model, where LRP1 is specifically deleted from GLAST-positive astrocyte precursor cells. The recombination with resulting knockout events was visualized by the simultaneous expression of the fluorescent reporter tdTomato. We observed a significantly increased number of GLT-1 expressing astrocytes in LRP1-depleted astrocytic cultures in comparison to control astrocytes. Furthermore, we investigated the influence of astrocytic LRP1 on neuronal activity and synaptogenesis using the co-culture of hippocampal neurons with control or LRP1-depleted astrocytes. These analyses revealed that the LRP1-deficient astrocytes caused a decreased number of single action potentials as well as a negatively influenced neuronal network activity. Moreover, the proportion of pre- and postsynaptic structures was significantly altered in neurons co-cultured with LPR1-depleted astrocytes. However, the number of structural synapses was not affected. Additionally, the supernatant of hippocampal neurons co-cultured with LRP1-deficient astrocytes showed an altered set of cytokines in comparison to the control condition, which potentially contributed to the altered neuronal transmission and synaptogenesis. Our results suggest astrocytic LRP1 as a modulator of synaptic transmission and synaptogenesis by altering the expression of the glutamate transporter on the cell surface on astrocytes and the release of cytokines in vitro.

TCEC Cup 7

This is the report of the 'TCEC Cup 7' computational experiment, the 7th TCEC knockout event and the second major part of TCEC’s 19th season. It started on 30th October 2020 and featured the best 32 available engines of the ‘TCEC 19’ league event. The games were held at Rapid tempo, the different format calling for different optimal settings in the engines and enabling a comparison with performance at slower tempi, albeit both being at super-GM level. STOCKFISH defeated LEELA CHESS ZERO in the Final. All decisive games in the supplementary data have been played out by FRITZ17 at search depth 24 to enable and to benchmark endgame practice.

TCEC Cup 4

This is a report of the 'TCEC Cup 4' computational experiment, the 4th TCEC knockout event and the second major part of TCEC’s 16th season. It started on 16th October 2019 and featured the best 32 engines of the ‘TCEC 16’ league event and the games were held at Blitz tempo, The different format called for different optimal settings in the engines, thereby enabling a comparison with performance at slower tempi. STOCKFISH retrieved the title from LEELA CHESS ZERO, defeating it when their perspectives diverted on move forty of game eight. All decisive games in the supplementary data have been played out by FRITZ17 at search depth 24 to enable and benchmark endgame practice.

TCEC Cup 5

This is the report of the 'TCEC Cup 5' computational experiment, the 5th TCEC knockout event and the second major part of TCEC’s 17th season. It started on 24th April 2020 and featured the best 16 available engines of the ‘TCEC 17’ league event. The games were held at Rapid tempo, the different format calling for different optimal settings in the engines. This enabled a comparison with performance at slower tempi although both are at super-GM level. STOCKFISH defeated LEELA CHESS ZERO in the Final. All decisive games in the supplementary data have been played out by FRITZ17 at search depth 24 to enable and to benchmark endgame practice.

TCEC Cup 3

This is a report of the 'TCEC Cup 3' computational experiment, the 3rd TCEC knockout event and the second major part of TCEC’s 15th season. It featured the best 32 engines of the ‘TCEC 15’ league event and the games were held at Blitz tempo. The different format called for different optimal settings in the engines, thereby enabling a comparison with performance at slower tempi. LEELA CHESS ZERO retained the TCEC Cup, defeating STOCKFISH this time. All decisive games in the supplementary data have been played out by FRITZ17 at search depth 24 to enable and benchmark endgame practice.

TCEC Cup 2

This is a report of the 'TCEC Cup 2' computational experiment, the second TCEC knockout event and the second major part of TCEC’s 14th season. It featured the best 32 engines of the ‘TCEC 14’ league event and the games were held at Blitz tempo. The different format called for different optimal settings in the engines, thereby enabling a comparison with performance at slower tempi. This event made history: the first major trophy win by a 'NN' Neural Network chess engine, LEELA CHESS ZERO, even though relatively disadvantaged by the faster tempo. All decisive games in the supplementary data have been played out by FRITZ17 at search depth 24 to benchmark endgame practice.

TCEC Cup 1

This is a report of the 'TCEC Cup 1' computational experiment, the first TCEC knockout event and the second major part of TCEC’s 13th season. It featured the best 32 engines of ‘TCEC 13’ and the games were held at Blitz tempo, The different format called for different optimal settings in the engines, thereby enabling a comparison with performance at slower tempi. This event notably featured the first appearance of two non-Shannon-AB, neural-network/MCTS engines, DEUS X and LEELA CHESS ZERO. LC0 notably was a quarter-finalist, having started as 21st seed. STOCKFISH beat HOUDINI in the final with the sole win, remarkably, a short and dramatic game. All decisive games in the supplementary data have been played out by FRITZ17 at search depth 24 to benchmark endgame practice.

The System Theoretic Accident Modelling and Process (STAMP) of medical pilot knock-out events: Pilot incapacitation and homicide-suicide

After the Germanwings flight 4U9525, mental health issues of pilots have been raised as a hazard to flight safety. Pilot homicide-suicide, as a special subtype of pilot incapacitation, can be examined at different levels of commercial aviation. We extended the System Theoretic Process Analysis (STPA), based on the System Theoretic Accident Modelling and Process (STAMP), to pilot behaviour to investigate how these knock-out events may be encountered in the whole system. Several safety actions have been identified to prevent hazardous pilot behaviour caused by medical incapacitation and homicidal-suicidal behaviour at different levels of the system. Pilot incapacitation is already handled very well in practice with respect to the regulations and procedures in current usage. In contrast, the prevention of pilot homicide-suicide is currently managed by aeromedical decision-making regulations and pilot support programs. The design of the flight deck compartment door and the balancing of privacy laws and public safety remain trade-offs amid a medical risk.

A Novel Tomato Fusarium Wilt Tolerance Gene.

The reduced mycorrhizal colonization (rmc) tomato mutant is unable to form mycorrhiza and is more susceptible to Fusarium wilt compared with its wild-type isogenic line 76R. The rmc mutant has a chromosomal deletion affecting five genes, one of which is similar to CYCLOPS. Loss of this gene is responsible for non-mycorrhizality in rmc but not enhanced Fusarium wilt susceptibility. Here, we describe assessment of a second gene in the rmc deletion, designated Solyc08g075770 that is expressed in roots. Sequence analyses show that Solyc08g075770 encodes a small transmembrane protein with putative phosphorylation and glycosylation sites. It is predicted to be localized in the plasma membrane and may function in transmembrane ion transport and/or as a cell surface receptor. Complementation and knock-out strategies were used to test its function. Some putative CRISPR/Cas-9 knock-out transgenic events exhibited Fusarium wilt susceptibility like rmc and some putative complementation lines were 76R-like, suggesting that the tomato Solyc08g075770 functions in Fusarium wilt tolerance. This is the first study to demonstrate that Solyc08g075770 is the contributor to the Tfw locus, conferring tolerance to Fusarium wilt in 76R which was lost in rmc.

GWAS signals revisited using human knockouts.

PurposeGenome-wide association studies (GWAS) have been instrumental to our understanding of the genetic risk determinants of complex traits. A common challenge in GWAS is the interpretation of signals, which are usually attributed to the genes closest to the polymorphic markers that display the strongest statistical association. Naturally occurring complete loss of function (knockout) of these genes in humans can inform GWAS interpretation by unmasking their deficiency state in a clinical context.MethodsWe exploited the unique population structure of Saudi Arabia to identify novel knockout events in genes previously highlighted in GWAS using combined autozygome/exome analysis.ResultsWe report five families with homozygous truncating mutations in genes that had only been linked to human disease through GWAS. The phenotypes observed in the natural knockouts for these genes (TRAF3IP2, FRMD3, RSRC1, BTBD9, and PXDNL) range from consistent with, to unrelated to, the previously reported GWAS phenotype.ConclusionWe expand the role of human knockouts in the medical annotation of the human genome, and show their potential value in informing the interpretation of GWAS of complex traits.

Collision-induced evaporation of water clusters and contribution of momentum transfer

The evaporation of water molecules from high-velocity argon atoms impinging on protonated water clusters has been computationally investigated using molecular dynamics simulations with the reactive OSS2 potential to model water clusters and the ZBL pair potential to represent their interaction with the projectile. Swarms of trajectories and an event-by-event analysis reveal the conditions under which a specific number of molecular evaporation events is found one nanosecond after impact, thereby excluding direct knockout events from the analysis. These simulations provide velocity distributions that exhibit two main features, with a major statistical component arising from a global redistribution of the collision energy into intermolecular degrees of freedom, and another minor but non-ergodic feature at high velocities. The latter feature is produced by direct impacts on the peripheral water molecules and reflects a more complete momentum transfer. These two components are consistent with recent experimental measurements and confirm that electronic processes are not explicitly needed to explain the observed non-ergodic behavior.

Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game.

Vaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccine-preventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers >20-fold and >50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine. Using a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to >50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases.