Knockout Mechanism Research Articles

CRISPRlnc: a machine learning method for lncRNA-specific single-guide RNA design of CRISPR/Cas9 system.

CRISPR/Cas9 is a promising RNA-guided genome editing technology, which consists of a Cas9 nuclease and a single-guide RNA (sgRNA). So far, a number of sgRNA prediction softwares have been developed. However, they were usually designed for protein-coding genes without considering that long non-coding RNA (lncRNA) genes may have different characteristics. In this study, we first evaluated the performances of a series of known sgRNA-designing tools in the context of both coding and non-coding datasets. Meanwhile, we analyzed the underpinnings of their varied performances on the sgRNA's specificity for lncRNA including nucleic acid sequence, genome location and editing mechanism preference. Furthermore, we introduce a support vector machine-based machine learning algorithm named CRISPRlnc, which aims to model both CRISPR knock-out (CRISPRko) and CRISPR inhibition (CRISPRi) mechanisms to predict the on-target activity of targets. CRISPRlnc combined the paired-sgRNA design and off-target analysis to achieve one-stop design of CRISPR/Cas9 sgRNAs for non-coding genes. Performance comparison on multiple datasets showed that CRISPRlnc was far superior to existing methods for both CRISPRko and CRISPRi mechanisms during the lncRNA-specific sgRNA design. To maximize the availability of CRISPRlnc, we developed a web server (http://predict.crisprlnc.cc) and made it available for download on GitHub.

Research progress and mitigation strategies for pod shattering resistance in rapeseed.

Mature rapeseed pods typically shatter when harvested, resulting in approximately 8-12% yield loss. Adverse weather conditions and mechanized harvesting can diminish pod yield by up to 50%, primarily owing to delays in harvesting and mechanical collisions. The pod shatter resistance index (PSRI) assesses pod damage. Recent research focused on comparing pod shatter resistance among varieties, evaluating methods, and studying gene knockout mechanisms. However, there remains a pressing need to broaden the scope of research. In particular, it is essential to recognize that pod shatter, a complex trait, influenced by genetics, environment, agronomic practices, and harvest techniques. Future studies should integrate these factors to develop comprehensive strategies to mitigate pod shatter, enhancing rapeseed yields and agricultural mechanization. This review explores factors affecting pod shatter resistance and strategies to improve it. Scoping literature review that adhered to the methodological framework for systematic reviews was performed using search engines such as Google Scholar, Web of Science, and the Chinese National Knowledge Infrastructure. This review aimed to identify pertinent articles, which were subsequently subjected to thorough screening and evaluation. The protocol for this literature review involved the following key steps: definition of research questions, development of a search strategy, development of data extraction strategy, synthesis of the extracted data, and organization and analysis of the extracted data. The review presents strategies for enhancing rapeseed yield during mechanized harvesting, focusing on four key areas: (i) selecting and breeding shatter-resistant varieties using DNA markers to establish a robust germplasm resource; (ii) optimizing cultivation technologies and agronomic measures to elicit favorable interactions between compact plant-type genotypes and the environment, thereby facilitating nutrient-related regulatory mechanisms of rapeseed pods to improve pod dry weight and resistance; (iii) innovating combine header design and structure to better suit rapeseed harvesting; and (iv) providing training for operators to enhance their harvesting skills. These comprehensive measures aim to minimize yield loss, increase production efficiency. To effectively reduce yield loss during mechanized harvesting of rapeseed, it is crucial to enhance resistance to pod shattering by addressing both internal physiological factors and external environmental conditions. This requires a holistic approach that includes genetic improvements, optimization of ecological conditions, careful cultivation management, and precise harvesting timing, along with ongoing research into traits related to machine harvesting to boost production efficiency and sustainability.

Direct double ionization for molecules of H2 and N2 induced by electron-impact

The direct double ionization of H2 and N2 molecules induced by electron-impact are investigated theoretically. Our theoretical calculation combines the binary-encounter-Bethe theory employed for molecular electron-impact single ionization and knock-out mechanism derived from the many-body perturbation theory. The process of electron-impact direct double ionization for molecules in our consideration is treated as the coupling of primary single ionization and knock-out process. The knock-out process is constituted by all the possible secondary single ionization processes. There is a good agreement between our theoretical results and available experimental measurements both for the molecules of H2 and N2. Our approach can obtain the cross section of each ionization channel, which is helpful to understanding for ionization competition mechanism of small homo-nuclear diatomic molecules.

Theoretical investigation of electron-impact double ionization of B-like C+ to Ne5+ ions

Electron-impact double ionization (DI) including direct and indirect processes of B-like ${\mathrm{C}}^{+}$ to ${\mathrm{Ne}}^{5+}$ ions is investigated using a two-step approach. The direct process is treated as a primary single ionization (SI) and subsequent knockout mechanism. The indirect process is considered as a primary SI of an inner-shell $1s$ electron followed by a single autoionization. Based on the two-step approach, the DI cross sections are calculated using flexible atomic code with distorted-wave approximation. The potential of the intermediate ion is approximately equivalent to the mean potential constructed by three ions involved for calculating the cross section of the direct process, while using the potential of the ionizing ion in each step of the present two-step approach overestimates results. The present cross sections agree with available theoretical and experimental data.

Shake-Off Process in Non-Sequential Single-Photon Double Ionization of Closed-Shell Atomic Targets

Amusia and Kheifets in 1984 introduced a Green’s function formalism to describe the effect of many-electron correlation on the ionization spectra of atoms. Here, we exploit this formalism to model the shake-off (SO) process, leading to the non-sequential single-photon two-electron ionization (double photoionization—DPI) of closed-shell atomic targets. We separate the SO process from another knock-out (KO) mechanism of DPI and show the SO prevalence away from the DPI threshold. We use this kinematic regime to validate our model by making a comparison with more elaborate techniques, such as convergent and time-dependent close coupling. We also use our model to evaluate the attosecond time delay associated with the SO process. Typically, the SO is very fast, taking only a few attoseconds to complete. However, it can take much longer in the DPI of strongly correlated systems, such as the H− ion as well as the subvalent shells of the Ar and Xe atoms and Cl− ion.

Multiple Auger decay following ionization-excitation of Ne i

The photoionization-excitation spectrum of Ne i, in which an electron in the $1s$ shell is ionized and the $2p$ orbital electron simultaneously excited to the $np$ orbital, is investigated in detail for complex satellite states ${\mathrm{Ne}}^{+}$ $1{s}^{\text{--}1}2{p}^{\text{--}1}{(}^{1,3}P)np$ $(n=3,4,5,6,7)$. Then, the multiple Auger decay process is studied using the multistep method, including cascade, knock-out, and shake-off mechanisms, based on perturbation theory for the satellite states ${\mathrm{Ne}}^{+}$ $1{s}^{\text{--}1}2{p}^{\text{--}1}({}^{1,3}\mathrm{P})np$ $(n=3,4)$ with two holes. The major single Auger decay can be described as a spectator process that valence electrons remain in the original orbitals. For double Auger decay, direct and cascade processes are considered, in which the direct mechanism is dominated by the spectator process, while the cascade mechanism is mainly from the participator process. For triple Auger decay, the important contribution is from the process consisting of direct double Auger and the subsequent single Auger electron emissions. The calculated ion yields of ${\mathrm{Ne}}^{2+}$, ${\mathrm{Ne}}^{3+}$, and ${\mathrm{Ne}}^{4+}$ for multiple Auger process of satellite states ${\mathrm{Ne}}^{+}$ $1{s}^{\text{--}1}2{p}^{\text{--}1}{(}^{1,3}P)np$ $(n=3,4)$ are consistent with experimental values [Hikosaka et al., Phys. Rev. A 97, 023405 (2018)].

Quasifree Photoionization under the Reaction Microscope

We experimentally investigated the quasifree mechanism (QFM) in one-photon double ionization of He and H2 at 800 eV photon energy and circular polarization with a COLTRIMS reaction microscope. Our work provides new insight into this elusive photoionization mechanism that was predicted by Miron Amusia more than four decades ago. We found the distinct four-fold symmetry in the angular emission pattern of QFM electrons from H2 double ionization that has previously only been observed for He. Furthermore, we provide experimental evidence that the photon momentum is not imparted onto the center of mass in quasifree photoionization, which is in contrast to the situation in single ionization and in double ionization mediated by the shake-off and knock-out mechanisms. This finding is substantiated by numerical results obtained by solving the system’s full-dimensional time-dependent Schrödinger equation beyond the dipole approximation.

Production of a Chimeric Mouse-Fish Monoclonal Antibody by the CRISPR/Cas9 Technology.

The CRISPR/Cas9 system, a defense mechanism naturally occurring in prokaryotes, has been recently repurposed as an RNA-guided DNA targeting platform and widely used as a powerful tool for genome editing. Here we describe how to modify the carboxy-terminal region, called Fragment crystallizable (Fc) region, of a murine monoclonal antibody by replacing the heavy chain constant exons with those from a teleost fish antibody by the CRISPR/Cas9 system. We outline optimal conditions for knockout and knockin mechanisms to edit the Immunoglobulin heavy chain (IgH) constant region gene locus in a murine hybridoma cell line. A chimeric mouse-fish monoclonal antibody can be successfully produced by hybridoma cell lines engineered according to this protocol.

Photofragmentation of large dimers into singly charged ions via the knockout mechanism

We study the fragmentation of very large dimers into two singly charged ions caused by absorption of a photon. In this process a photo electron emitted from one atom of the dimer has certain chances to hit the other atom knocking out one of its electrons. This results in the production of two singly charged ions and the consequent Coulomb explosion of the residual doubly charged system. We develop a theory of this process and apply it to calculate the fragmentation of $^4$He$_2$, $^7$Li - $^4$He and $^6$Li - $^4$He dimers. Our results for the helium dimer are in good agreement with available experimental data. For the Li-He dimers (where experimental data are still absent) our results predict that this fragmentation mechanism becomes much more efficient than in the case of He$_2$. We also show that, provided the recoil effects are of minor importance, the so called reflection approximation is very accurate also for large dimers.

Electron-impact single and double ionization of Fe12+

The cross sections of electron-impact single and double ionization of Fe12+ are investigated using the distorted-wave (DW) method and two-step approximate formula based on the knockout (KO) mechanism. Both direct and indirect processes are studied in single and double electron-impact ionization. The better agreements between our theoretical results and experiment measurements are obtained contrasted to earlier theoretical works. Adequate indirect ionization channels with larger configurations space are used in our calculation, which is significant for understanding of the mechanism of single and double electron-impact ionization for highly charge ion Fe12+.

Competition between the shake-off and knockout mechanisms in the double and triple photoionization of the halothane molecule (C2HBrClF3).

The ratios of single, double, and triple ionizations to the total photoionization of the halothane (C2HBrClF3) molecule have been investigated by a single-photon ionization in the energy range from 21.21 eV to 320 eV. In the valence region, the multiple ionization results can be described by a sum of contributions generated from the shake-off and the two-step one models. At low photon energies (from the threshold of triple ionization up to 100 eV), the triple photoionization dynamics of halothane can be reasonably well described by a model involving a classical electron impact double ionization of the singly ionized parent ion.

Theoretical investigation of electron-impact multiple ionization of O ii-iv ions

The electron-impact double ionization (DI) and triple ionization (TI) for O ii-iv ions are investigated using multistep approaches within the framework of many-body perturbation theory. The indirect DI and TI are treated as the single ionization of an inner-shell $1s$ electron followed by the single and double Auger decays, respectively. The knockout mechanism is employed to deal with the direct ionization process. The present approaches reproduce the available experimental cross sections for the DI of O ii-iv ions with much better accuracy compared to previous calculations. The TI cross sections of the O ii ion are calculated, which are in good agreement with the recent experimental data [Lecointre et al., J. Phys. B 46, 205201 (2013)]. It is found that the electron correlation effect and the energy distribution of the intermediate electron, which is taken as the incident electron of the subsequent knockout process, play an important role in the multistep treatment of the multiple ionization process.

Dominance of double processes in complete Auger decay of Rb+(3d−1)

The complete Auger decay of ${\mathrm{Rb}}^{+}(3{d}^{\ensuremath{-}1})$ including single and double processes is investigated using the distorted wave approximation. The direct double Auger decay was calculated by separating the knock-out and shake-off mechanisms. For single Auger decay of the $3{d}^{\ensuremath{-}1}$ hole state, it is generally believed that the dominant channels should be levels belonging to the configurations of $[1{s}^{2}2{s}^{2}2{p}^{6}3{s}^{2}3{p}^{6}3{d}^{10}]4{s}^{2}4{p}^{4}5s$, $4s4{p}^{5}5s$, and $4{s}^{0}4{p}^{6}5s$. Here we predict that the strongest channels originate from $4{s}^{2}4{p}^{3}4d5s$, which accounts for 36.3% of the single Auger decay rate. The levels belonging to this configuration can further decay to ${\mathrm{Rb}}^{3+}$, resulting in a large fraction of cascade double Auger decay. Moreover, the probability of direct double Auger decay in Rb is also high because of its $5s$ electron. The branching ratios of cascade and direct double Auger decay are predicted to be 50.60% and 22.13%, respectively, resulting in the dominance of double Auger decay with a fraction of 72.73% in the complete Auger decay of ${\mathrm{Rb}}^{+}(3{d}^{\ensuremath{-}1})$.

Novel design for the adjustable knock-out mechanism with a Roberts linkage

This article presents a new adjustable knock-out mechanism of a bolt former with a Roberts linkage and an adjustable crank-rocker linkage. The design requirements of the new mechanism are based on the analysis of an existing adjustable knock-out mechanism. The new adjustable knock-out mechanism mainly consisting of a Roberts linkage, and an adjustable crank-rocker linkage is proposed to meet the specified requirements. The path of the knock-out screw is approximately a straight line and the method presented is illustrated by an example.

Charged Particle Production in Stopped Pion Absorption by Nuclei

The spectra of 3,4He charged particles produced in stopped pion absorption by nuclei were measured for 17 nuclei in the mass-number range of 6 ≤ A ≤ 209 by means of a semiconductor spectrometer. These data were analyzed on the basis of a phenomenological model where the absorption reaction in question is treated as a three-stage process: primary absorption by intranuclear clusters, preequilibrium stage, and evaporation stage. It is shown that a dominant contribution to the production of helium isotopes with energies not less than 20 MeV comes from the preequilibrium stage. The contribution of the knockout and pickup mechanisms to 3,4He production is estimated.

An edible fungi Pleurotus ostreatus inhibits adipogenesis via suppressing expression of PPAR γ and C/EBP α in 3T3-L1 cells: In vitro validation of gene knock out of RNAs in PPAR γ using CRISPR spcas9

ObjectiveObesity is now well recognized as a disorder, one that is essentially preventable through changes in lifestyle. Obesity is also a main concern associated with expanded morbidity and mortality from many noncommunicable illnesses (NCDs). The study aimed to determine the antiobesity effect of Pleurotus ostreatus (PO) and its bioactive anthraquinone (AQ). The overall promoter genes CEBPα (CCAAT enhancer binding protein α) and PPARγ (Peroxisome proliferator activated receptor γ) in controlling the homeostasis of glucose was analysed using 3T3-L1 cell line. Finally, an insilico study was carried out using CRISPR software to identify the RNA’s involved in adipogenesis especially of the control gene PPARγ. Materials and methodsPreliminary screening of the edible fungi and their bio actives led to the marvellous discovery of side effect free agonists for treating obesity (adipogenesis). An edible fungi Pleurotus ostreatus (PO) were analysed in a screening platform with different series of tests for adipocyte differentiation, triglyceride analysis, lipolysis determination, glucose uptake assay, cytotoxicity assay and lipase activity followed by specific gene expression analysis. The gene knockout mechanism was also elucidated by CRISPR spcas 9 tool. ResultsThe antiadipogenic (antiobesity) activity of DMSO extract of PO were found to stimulate the insulin dependent uptake of glucose. The extract also decreased the levels of triglycerides and glycerol accumulation in differentiated adipocyte cells. The binding FABP4 (Fatty acid binding protein) and transport protein FATP1 (Fatty acid transport protein) along with the fat breaking LPL (lipoprotein lipase) was found to be inhibited after the PO treatment at varying concentration (0–300 μg/ml). CRISPR spcas9 genome editing software was used as an insilico approach in validating the efficiency of mouse embryonic and human adipogenic cell line (3T3-L1). These tool analysed and found 4 RNAs gene knock out possibilities in PPARγ and their efficiency for further treating obesity. ConclusionThese novel finding contribute to the confirmation that edible fungi PO and it’s bioactive AQ is an adequate supplement for constraining the lipid and triglycerides in differentiated mature adipocytes by reversing the fat deposition. Thereby, forbidding the enzymes linked with fat absorption. Besides, the CRISPR tool identified gene knock out possibilities of control gene PPARγ, will pave a way in further research for treating obesity.

Single and double Auger decay of 4f-ionized mercury including cascade and direct processes**Project supported by the National Key Research and Development Program of China (Grant No. 2017YFA0402300), the National Natural Science Foundation of China (Grant Nos. 11774344 and 11474033), and the Joint Foundation of the National Natural Science Foundation and the China Academy of Engineering Physics (Grant No. U1330117).

Single (SA) and double (DA) Auger decay including cascade and direct processes are investigated for Hg 4f−1 with multiconfiguration Dirac–Fock method and two-step approaches, i.e., knockout and shakeoff mechanisms. Due to the computational effort, only the major transitions are considered to describe the SA and DA decays for the Hg+ ions with complex electronic configurations. In order to estimate the Auger transition energies and amplitudes, the reference configuration sets producing the configuration state functions are carefully chosen for balancing electron correlations among the successive singly, doubly and triply ionized mercury. The Auger rates and electron spectra, DA probabilities as well as the populations of the final Hg3+ states are obtained. Our results well explain the recent experimental data about the 4f hole states of Hg [Palaudoux J et al., Phys. Rev. A 91 012513 (2015)], and could provide guidance for further studies on complex atoms. Particularly for the DA decay, the contributions of the direct processes, which are neglected in their calculations, are found to be important, accounting for as high as about 38% and 34% of the total DA decays for the and , respectively.

Auger decay including direct double processes of K-shell hollow states of Ne+ and the related hypersatellite radiative transitions

In this study, we theoretically investigated single and double Auger decay processes from the K−2V (V = 3s, 3p, 3d, 4s, 4p, 4d) hollow states of Ne+ in the framework of the first- and second-order perturbation theory implemented by the distorted wave approximation. The direct double Auger decay rates were calculated based on the separation of knock-out and shake-off mechanisms. Atomic data, including the transition energy, single and double Auger decay rates and natural lifetime width, are obtained and compared with the experimental and theoretical results available in the literature. The natural lifetime widths of the K−2V hollow states, including the contributions from single and direct double Auger decay, are in excellent agreement with a recent experiment. Using our theoretical results, we diagnosed the relative population fractions of the initial quantum states prepared in the experiment and interpreted the measured Auger spectra. There is an excellent agreement between the theoretical and experimental results. Finally, the hypersatellite Kα radiative transitions relevant to these hollow states were investigated using the natural lifetime widths determined by single and direct double Auger rates.

A practical theoretical formalism for atomic multielectron processes: direct multiple ionization by a single auger decay or by impact of a single electron or photon

Multiple electron processes occur widely in atoms, molecules, clusters, and condensed matters when they are interacting with energetic particles or intense laser fields. Direct multielectron processes (DMEP) are the most complicated among the general multiple electron processes and are the most difficult to describe theoretically. In this work, a unified and accurate theoretical formalism is proposed on the DMEP of atoms including the multiple auger decay and multiple ionization by an impact of a single electron or a single photon based on the atomic collision theory described by a correlated many-body Green's function. Such a practical treatment is made possible by taking consideration of the different coherence features of the atoms (matter waves) in the initial and final states. We first explain how the coherence characteristics of the ejected continuum electrons is largely destructed, by taking the electron impact direct double ionization process as an example. The direct double ionization process is completely different from the single ionization where the complete interference can be maintained. The detailed expressions are obtained for the energy correlations among the continuum electrons and energy resolved differential and integral cross sections according to the separation of knock-out (KO) and shake-off (SO) mechanisms for the electron impact direct double ionization, direct double and triple auger decay, and double and triple photoionization (TPI) processes. Extension to higher order DMEP than triple ionization is straight forward by adding contributions of the following KO and SO processes. The approach is applied to investigate the electron impact double ionization processes of C+, N+, and O+, the direct double and triple auger decay of the K-shell excited states of C+ and , and the double and TPI of lithium. Comparisons with the experimental and other theoretical investigations wherever available in the literature show that our theoretical formalism is accurate and effective in treating the atomic multielectron processes.

Multiple Auger decay probabilities of neon from the 1s -core-hole state

The multiple Auger decays of the $\mathrm{Ne}\phantom{\rule{0.28em}{0ex}}1{s}^{\ensuremath{-}1}$ state including double and triple Auger processes are investigated within the framework of perturbation theory. The contributions of the cascade and direct processes are determined for the double Auger decay. In the cascade processes, the choice of the orbital sets and configuration interaction can strongly affect the partial probabilities for the specific configurations of $\mathrm{N}{\mathrm{e}}^{3+}$. The multistep approaches, i.e., the knockout and shakeoff mechanisms, are implemented to deal with the direct double Auger processes for which the total and partial probabilities corresponding to specific configurations of $\mathrm{N}{\mathrm{e}}^{3+}$ are calculated and reveal that the knockout is dominant. Finally, the probabilities of the triple Auger decays that are decomposed into a double Auger process and a subsequent emission of a single electron are obtained using the cascade and knockout mechanisms. The calculated probabilities agree reasonably well with the available experimental data.