CRISPR-Cas Systems and Genome Editing: Beginning the Era of CRISPR/Cas Therapies for Humans.

Harnessing of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) systems for detection, chemical modification, and sequence editing of nucleic acids dramatically changed many fields of fundamental science, biotechnology, and biomedicine [...].

Synergistic Effect of a Combination of Proteasome and Ribonucleotide Reductase Inhibitors in a Biochemical Model of the Yeast Saccharomyces cerevisiae and a Glioblastoma Cell Line.

Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors.

Engineering Cas9: next generation of genomic editors.

The Cas9 endonuclease of the CRISPR/Cas type IIA system from Streptococcus pyogenes is the heart of genome editing technology that can be used to treat human genetic and viral diseases. Despite its large size and other drawbacks, S. pyogenes Cas9 remains the most widely used genome editor. A vast amount of research is aimed at improving Cas9 as a promising genetic therapy. Strategies include directed evolution of the Cas9 protein, rational design, and domain swapping. The first generation of Cas9 editors comes directly from the wild-type protein. The next generation is obtained by combining mutations from the first-generation variants, adding new mutations to them, or refining mutations. This review summarizes and discusses recent advances and ways in the creation of next-generation genomic editors derived from S. pyogenes Cas9. KEY POINTS: • The next-generation Cas9-based editors are more active than in the first one. • PAM-relaxed variants of Cas9 are improved by increased specificity and activity. • Less mutagenic and immunogenic variants of Cas9 are created.

Enhanced Piezoelectric Response at Nanoscale Vortex Structures in Ferroelectrics.

The piezoelectric response is a measure of the sensitivity of a material's polarization to stress or its strain to an applied field. Using in operando X-ray Bragg coherent diffraction imaging, we observe that topological vortices are the source of a 5-fold enhancement of the piezoelectric response near the vortex core. The vortices form where several low-symmetry ferroelectric phases and phase boundaries coalesce. Unlike bulk ferroelectric solid solutions in which a large piezoelectric response is associated with coexisting phases in the proximity of the triple point, the largest responses for pure BaTiO3 at the nanoscale are in spatial regions of extremely small spontaneous polarization at vortex cores. The response decays inversely with polarization away from the vortex, analogous to the behavior in bulk ceramics as the cation compositions are varied away from the triple point. We use first-principles-based molecular dynamics to augment our observations, and our results suggest that nanoscale piezoelectric materials with a large piezoelectric response can be designed within a parameter space governed by vortex cores. Our findings have implications for the development of next-generation nanoscale piezoelectric materials.

Challenges of CRISPR/Cas-Based Cell Therapy for Type 1 Diabetes: How Not to Engineer a "Trojan Horse".

Type 1 diabetes mellitus (T1D) is an autoimmune disease caused by the destruction of insulin-producing ß-cells in the pancreas by cytotoxic T-cells. To date, there are no drugs that can prevent the development of T1D. Insulin replacement therapy is the standard care for patients with T1D. This treatment is life-saving, but is expensive, can lead to acute and long-term complications, and results in reduced overall life expectancy. This has stimulated the research and development of alternative treatments for T1D. In this review, we consider potential therapies for T1D using cellular regenerative medicine approaches with a focus on CRISPR/Cas-engineered cellular products. However, CRISPR/Cas as a genome editing tool has several drawbacks that should be considered for safe and efficient cell engineering. In addition, cellular engineering approaches themselves pose a hidden threat. The purpose of this review is to critically discuss novel strategies for the treatment of T1D using genome editing technology. A well-designed approach to ß-cell derivation using CRISPR/Cas-based genome editing technology will significantly reduce the risk of incorrectly engineered cell products that could behave as a "Trojan horse".

Culturable Bacterial Endophytes of Wild White Poplar (Populus alba L.) Roots: A First Insight into Their Plant Growth-Stimulating and Bioaugmentation Potential.

The white poplar (Populus alba L.) has good potential for a green economy and phytoremediation. Bioaugmentation using endophytic bacteria can be considered as a safe strategy to increase poplar productivity and its resistance to toxic urban conditions. The aim of our work was to find the most promising strains of bacterial endophytes to enhance the growth of white poplar in unfavorable environmental conditions. To this end, for the first time, we performed whole-genome sequencing of 14 bacterial strains isolated from the tissues of the roots of white poplar in different geographical locations. We then performed a bioinformatics search to identify genes that may be useful for poplar growth and resistance to environmental pollutants and pathogens. Almost all endophytic bacteria obtained from white poplar roots are new strains of known species belonging to the genera Bacillus, Corynebacterium, Kocuria, Micrococcus, Peribacillus, Pseudomonas, and Staphylococcus. The genomes of the strains contain genes involved in the enhanced metabolism of nitrogen, phosphorus, and metals, the synthesis of valuable secondary metabolites, and the detoxification of heavy metals and organic pollutants. All the strains are able to grow on media without nitrogen sources, which indicates their ability to fix atmospheric nitrogen. It is concluded that the strains belonging to the genus Pseudomonas and bacteria of the species Kocuria rosea have the best poplar growth-stimulating and bioaugmentation potential, and the roots of white poplar are a valuable source for isolation of endophytic bacteria for possible application in ecobiotechnology.

ASCL1 Is Involved in the Pathogenesis of Schizophrenia by Regulation of Genes Related to Cell Proliferation, Neuronal Signature Formation, and Neuroplasticity.

Schizophrenia (SZ) is a common psychiatric neurodevelopmental disorder with a complex genetic architecture. Genome-wide association studies indicate the involvement of several transcription factors, including ASCL1, in the pathogenesis of SZ. We aimed to identify ASCL1-dependent cellular and molecular mechanisms associated with SZ. We used Capture-C, CRISPR/Cas9 systems and RNA-seq analysis to confirm the involvement of ASCL1 in SZ-associated pathogenesis, establish a mutant SH-SY5Y line with a functional ASCL1 knockout (ASCL1-del) and elucidate differentially expressed genes that may underlie ASCL1-dependent pathogenic mechanisms. Capture-C confirmed the spatial interaction of the ASCL1 promoter with SZ-associated loci. Transcriptome analysis showed that ASCL1 regulation may be through a negative feedback mechanism. ASCL1 dysfunction affects the expression of genes associated with the pathogenesis of SZ, as well as bipolar and depressive disorders. Genes differentially expressed in ASCL1-del are involved in cell mitosis, neuronal projection, neuropeptide signaling, and the formation of intercellular contacts, including the synapse. After retinoic acid (RA)-induced differentiation, ASCL1 activity is restricted to a small subset of genes involved in neuroplasticity. These data suggest that ASCL1 dysfunction promotes SZ development predominantly before the onset of neuronal differentiation by slowing cell proliferation and impeding the formation of neuronal signatures.

Yeast Ribonucleotide Reductase Is a Direct Target of the Proteasome and Provides Hyper Resistance to the Carcinogen 4-NQO.

Various external and internal factors damaging DNA constantly disrupt the stability of the genome. Cells use numerous dedicated DNA repair systems to detect damage and restore genomic integrity in a timely manner. Ribonucleotide reductase (RNR) is a key enzyme providing dNTPs for DNA repair. Molecular mechanisms of indirect regulation of yeast RNR activity are well understood, whereas little is known about its direct regulation. The study was aimed at elucidation of the proteasome-dependent mechanism of direct regulation of RNR subunits in Saccharomyces cerevisiae. Proteome analysis followed by Western blot, RT-PCR, and yeast plating analysis showed that upregulation of RNR by proteasome deregulation is associated with yeast hyper resistance to 4-nitroquinoline-1-oxide (4-NQO), a UV-mimetic DNA-damaging drug used in animal models to study oncogenesis. Inhibition of RNR or deletion of RNR regulatory proteins reverses the phenotype of yeast hyper resistance to 4-NQO. We have shown for the first time that the yeast Rnr1 subunit is a substrate of the proteasome, which suggests a common mechanism of RNR regulation in yeast and mammals.

Decoupling the Contributions of Different Instability Mechanisms to the PEMFC Performance Decay of Non-noble Metal O2-Reduction Catalysts.

Non-noble metal catalysts (NNMCs) hold the potential to replace the expensive Pt-based materials currently used to speed up the oxygen reduction reaction (ORR) in proton exchange membrane fuel cell (PEMFC) cathodes, but they feature poor durability that inhibits their implementation in commercial PEMFCs. This performance decay is commonly ascribed to the operative demetallation of their ORR-active sites, the electro-oxidation of the carbonaceous matrix that hosts these active centers, and/or the chemical degradation of the ionomer, active sites, and/or carbon support by radicals derived from the H2O2 produced as an ORR by-product. However, little is known regarding the relative contributions of these mechanisms to the overall PEMFC performance loss. With this motivation, in this study, we combined four degradation protocols entailing different cathode gas feeds (i.e., air vs N2), potential hold values, and durations to decouple the relative impact of the above deactivation mechanisms to the overall performance decay. Our results indicate that H2O2-related instability does not depend on the operative voltage but only on the ORR charge. Moreover, the electro-oxidation of the carbon matrix at high potentials (which for the catalyst tested herein triggers at 0.7 V) seems to be more detrimental to the NNMCs' activity than the demetallation occurring at low potentials.

Improving the on-target activity of high-fidelity Cas9 editors by combining rational design and random mutagenesis.

Genomic and post-genomic editors based on CRISPR/Cas systems are widely used in basic research and applied sciences, including human gene therapy. Most genome editing tools are based on the CRISPR/Cas9 type IIA system from Streptococcus pyogenes. Unfortunately, a number of drawbacks have hindered its application in therapeutic approaches, the most serious of which is the relatively high level of off-targets. To overcome this obstacle, various high-fidelity Cas9 variants have been created. However, they show reduced on-target activity compared to wild-type Cas9 possibly due to increased sensitivity to eukaryotic chromatin. Here, we combined a rational approach with random mutagenesis to create a set of new Cas9 variants showing high specificity and increased activity in Saccharomyces cerevisiae yeast. Moreover, a novel mutation in the PAM (protospacer adjacent motif)-interacting Cas9 domain was found, which increases the on-target activity of high-fidelity Cas9 variants while retaining their high specificity. The obtained data suggest that this mutation acts by weakening the eukaryotic chromatin barrier for Cas9 and rearranging the RuvC active center. Improved Cas9 variants should further advance genome and post-genome editing technologies. KEY POINTS: • D147Y and P411T mutations increase the activity of high-fidelity Cas9 variants. • The new L1206P mutation further increases the activity of high-fidelity Cas9 variants. • The L1206P mutation weakens the chromatin barrier for Cas9 editors.

Increasing the Activity of the High-Fidelity SpyCas9 Form in Yeast by Directed Mutagenesis of the PAM-Interacting Domain.

CRISPR/Cas systems are used for genome editing, both in basic science and in biotechnology. However, CRISPR/Cas editors have several limitations, including insufficient specificity leading to "off-targets" and the dependence of activity on chromatin state. A number of highly specific Cas9 variants have now been obtained, but most of them are characterized by reduced activity on eukaryotic chromatin. We identified a spatial cluster of amino acid residues in the PAM-recognizing domain of Streptococcus pyogenes Cas9, whose mutations restore the activity of one of the highly specific forms of SpyCas9 without reducing its activity in Saccharomyces cerevisiae. In addition, one of these new mutations also increases the efficiency of SpyCas9-mediated editing of a site localized on the stable nucleosome. The improved Cas9 variants we obtained, which are capable of editing hard-to-reach regions of the yeast genome, may help in both basic research and yeast biotechnological applications.

Probing the Cellular Fate of the Protein Corona around Nanoparticles with Nanofocused X-ray Fluorescence Imaging.

X-ray fluorescence imaging (XRF-imaging) with subcellular resolution is used to study the intracellular integrity of a protein corona that was pre-formed around gold nanoparticles (AuNP). Artificial proteins engineered to obtain Gd coordination for detection by XRF-imaging were used to form the corona. Indications about the degradation of this protein corona at a cellular and subcellular level can be observed by following the Au and Gd quantities in a time and spatial-dependent manner. The extended acquisition times necessary for capturing individual XRF-imaging cell images result in relatively small sample populations, stressing the need for faster image acquisition strategies in future XRF-imaging-based studies to deal with the inherent variability between cells. Still, results obtained reveal degradation of the protein corona during cellular trafficking, followed by differential cellular processing for AuNP and Gd-labelled proteins. Overall, this demonstrates that the dynamic degradation of the protein corona can be tracked by XRF-imaging to a certain degree.

Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems.

Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.

Role of Polyamine-Induced Dimerization of Antizyme in Its Cellular Functions.

The polyamines, spermine (Spm) and spermidine (Spd), are important for cell growth and function. Their homeostasis is strictly controlled, and a key downregulator of the polyamine pool is the polyamine-inducible protein, antizyme 1 (OAZ1). OAZ1 inhibits polyamine uptake and targets ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, for proteasomal degradation. Here we report, for the first time, that polyamines induce dimerization of mouse recombinant full-length OAZ1, forming an (OAZ1)2-Polyamine complex. Dimerization could be modulated by functionally active C-methylated spermidine mimetics (MeSpds) by changing the position of the methyl group along the Spd backbone-2-MeSpd was a poor inducer as opposed to 1-MeSpd, 3-MeSpd, and Spd, which were good inducers. Importantly, the ability of compounds to inhibit polyamine uptake correlated with the efficiency of the (OAZ1)2-Polyamine complex formation. Thus, the (OAZ1)2-Polyamine complex may be needed to inhibit polyamine uptake. The efficiency of polyamine-induced ribosomal +1 frameshifting of OAZ1 mRNA could also be differentially modulated by MeSpds-2-MeSpd was a poor inducer of OAZ1 biosynthesis and hence a poor downregulator of ODC activity unlike the other MeSpds. These findings offer new insight into the OAZ1-mediated regulation of polyamine homeostasis and provide the chemical tools to study it.

Yugra State University Biological Collection (Khanty-Mansiysk, Russia): general and digitisation overview.

BACKGROUND: The history of biological collections and digitisation initiatives in northern West Siberia is relatively new due to recent development of the region. The Center for Biodiversity Data Mobilization was established to promote the initiative, led by the Yugra State University. This organisation itself has a relatively young collection of biological specimens, which was, until recently, in a disintegrated state and only partly mobilised. The Yugra State University Biological Collection (YSU BC) currently includes three subdivisions differring by history and taxonomic groups, but also by details of management and storage conditions: the Fungarium, the Bryological collection and the Herbarium collection of YSU.The paper describes the general structure of the Yugra State University Biological Collection, its history, storage conditions, management practices, geographical, temporal and taxonomical coverage. The paper is underlined by three datasets of the collections databases published in GBIF, which are described in detail. The databases are managed in Specify 6 and 7 software and accessed through Specify Web Portal and through GBIF. NEW INFORMATION: The Yugra State University Biological Collection made an active reorganisation of physical storage conditions and data management recently, providing the model for other collections in the region. This paper describes the history, general structure, management practices and data management of all three parts of this collection for the first time.Although one part of the collection (Fungarium YSU) was mobilised earlier, last year, we mobilised data of the Bryological and Vascular plants (Herbarium) collections. The three datasets of the corresponding collections in GBIF were increased by about 6000 georeferenced records during the last year.

CRISPR/Cas-Based Approaches to Study Schizophrenia and Other Neurodevelopmental Disorders.

The study of diseases of the central nervous system (CNS) at the molecular level is challenging because of the complexity of neural circuits and the huge number of specialized cell types. Moreover, genomic association studies have revealed the complex genetic architecture of schizophrenia and other genetically determined mental disorders. Investigating such complex genetic architecture to decipher the molecular basis of CNS pathologies requires the use of high-throughput models such as cells and their derivatives. The time is coming for high-throughput genetic technologies based on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas systems to manipulate multiple genomic targets. CRISPR/Cas systems provide the desired complexity, versatility, and flexibility to create novel genetic tools capable of both altering the DNA sequence and affecting its function at higher levels of genetic information flow. CRISPR/Cas tools make it possible to find and investigate the intricate relationship between the genotype and phenotype of neuronal cells. The purpose of this review is to discuss innovative CRISPR-based approaches for studying the molecular mechanisms of CNS pathologies using cellular models.

A Strategy for the Rapid Development of a Safe Vibrio cholerae Candidate Vaccine Strain.

Approximately 1/6 of humanity is at high risk of experiencing cholera epidemics. The development of effective and safe vaccines against Vibrio cholerae, the primary cause of cholera, is part of the public health measures to prevent cholera epidemics. Natural nontoxigenic V. cholerae isolates represent a source of new genetically improved and relatively safe vaccine strains. However, the genomic engineering of wild-type V. cholerae strains is difficult, and these strains are genetically unstable due to their high homologous recombination activity. We comprehensively characterized two V. cholerae isolates using genome sequencing, bioinformatic analysis, and microscopic, physiological, and biochemical tests. Genetic constructs were Gibson assembled and electrotransformed into V. cholerae. Bacterial colonies were assessed using standard microbiological and immunological techniques. As a result, we created a synthetic chromoprotein-expressing reporter operon. This operon was used to improve the V. cholerae genome engineering approach and monitor the stability of the genetic constructs. Finally, we created a stable candidate V. cholerae vaccine strain bearing a recA deletion and expressing the ß-subunit of cholera toxin. Thus, we developed a strategy for the rapid creation of genetically stable and relatively safe candidate vaccine strains. This strategy can be applied not only to V. cholerae but also to other important human bacterial pathogens.

A Multi-Scale and Multi-Technique Approach for the Characterization of the Effects of Spatially Fractionated X-ray Radiation Therapies in a Preclinical Model.

The purpose of this study is to use a multi-technique approach to detect the effects of spatially fractionated X-ray Microbeam (MRT) and Minibeam Radiation Therapy (MB) and to compare them to seamless Broad Beam (BB) irradiation. Healthy- and Glioblastoma (GBM)-bearing male Fischer rats were irradiated in-vivo on the right brain hemisphere with MRT, MB and BB delivering three different doses for each irradiation geometry. Brains were analyzed post mortem by multi-scale X-ray Phase Contrast Imaging-Computed Tomography (XPCI-CT), histology, immunohistochemistry, X-ray Fluorescence (XRF), Small- and Wide-Angle X-ray Scattering (SAXS/WAXS). XPCI-CT discriminates with high sensitivity the effects of MRT, MB and BB irradiations on both healthy and GBM-bearing brains producing a first-time 3D visualization and morphological analysis of the radio-induced lesions, MRT and MB induced tissue ablations, the presence of hyperdense deposits within specific areas of the brain and tumor evolution or regression with respect to the evaluation made few days post-irradiation with an in-vivo magnetic resonance imaging session. Histology, immunohistochemistry, SAXS/WAXS and XRF allowed identification and classification of these deposits as hydroxyapatite crystals with the coexistence of Ca, P and Fe mineralization, and the multi-technique approach enabled the realization, for the first time, of the map of the differential radiosensitivity of the different brain areas treated with MRT and MB. 3D XPCI-CT datasets enabled also the quantification of tumor volumes and Ca/Fe deposits and their full-organ visualization. The multi-scale and multi-technique approach enabled a detailed visualization and classification in 3D of the radio-induced effects on brain tissues bringing new essential information towards the clinical implementation of the MRT and MB radiation therapy techniques.

Cellular Models in Schizophrenia Research.

Schizophrenia (SZ) is a prevalent functional psychosis characterized by clinical behavioural symptoms and underlying abnormalities in brain function. Genome-wide association studies (GWAS) of schizophrenia have revealed many loci that do not directly identify processes disturbed in the disease. For this reason, the development of cellular models containing SZ-associated variations has become a focus in the post-GWAS research era. The application of revolutionary clustered regularly interspaced palindromic repeats CRISPR/Cas9 gene-editing tools, along with recently developed technologies for cultivating brain organoids in vitro, have opened new perspectives for the construction of these models. In general, cellular models are intended to unravel particular biological phenomena. They can provide the missing link between schizophrenia-related phenotypic features (such as transcriptional dysregulation, oxidative stress and synaptic dysregulation) and data from pathomorphological, electrophysiological and behavioural studies. The objectives of this review are the systematization and classification of cellular models of schizophrenia, based on their complexity and validity for understanding schizophrenia-related phenotypes.

CRISPR/Cas9-Mediated Genome Engineering Reveals the Contribution of the 26S Proteasome to the Extremophilic Nature of the Yeast Debaryomyces hansenii.

The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo- and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single- and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows high-efficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno- and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.