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.

Editing Metabolism, Sex, and Microbiome: How Can We Help Poplar Resist Pathogens?

Poplar (Populus) is a genus of woody plants of great economic value. Due to the growing economic importance of poplar, there is a need to ensure its stable growth by increasing its resistance to pathogens. Genetic engineering can create organisms with improved traits faster than traditional methods, and with the development of CRISPR/Cas-based genome editing systems, scientists have a new highly effective tool for creating valuable genotypes. In this review, we summarize the latest research data on poplar diseases, the biology of their pathogens and how these plants resist pathogens. In the final section, we propose to plant male or mixed poplar populations; consider the genes of the MLO group, transcription factors of the WRKY and MYB families and defensive proteins BbChit1, LJAMP2, MsrA2 and PtDef as the most promising targets for genetic engineering; and also pay attention to the possibility of microbiome engineering.

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".

Multifaceted Nothobranchius.

Annual killifish of the genus Nothobranchius are seeing a rapid increase in scientific interest over the years. A variety of aspects surrounding the egg-laying Cyprinodontiformes is being extensively studied, including their aging. Inhabiting drying water bodies of Africa rarely allows survival through more than one rainy season for the Nothobranchius populations. Therefore, there is no lifespan-related bias in natural selection, which has ultimately led to the decreased efficiency of DNA repair system. Aging of the Nothobranchius species is studied both under normal conditions and under the influence of potential geroprotectors, as well as genetic modifications. Most biogerontological studies are conducted using the species Nothobranchius furzeri (GRZ isolate), which has a lifespan of 3 to 7 months. However, the list of model species of Nothobranchius is considerably wider, and the range of advanced research areas with their participation extends far beyond gerontology. This review summarizes the most interesting and promising topics developing in the studies of the fish of Nothobranchius genus. Both classical studies related to lifespan control and rather new ones are discussed, including mechanisms of diapause, challenges of systematics and phylogeny, evolution of sex determination mechanisms, changes in chromosome count, occurrence of multiple repeated DNA sequences in the genome, cognitive and behavioral features and social stratification, as well as methodological difficulties in working with Nothobranchius.

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.

Heat stress analysis suggests a genetic basis for tolerance in Macrocystis pyrifera across developmental stages.

Kelps are vital for marine ecosystems, yet the genetic diversity underlying their capacity to adapt to climate change remains unknown. In this study, we focused on the kelp Macrocystis pyrifera a species critical to coastal habitats. We developed a protocol to evaluate heat stress response in 204 Macrocystis pyrifera genotypes subjected to heat stress treatments ranging from 21 °C to 27 °C. Here we show that haploid gametophytes exhibiting a heat-stress tolerant (HST) phenotype also produced greater biomass as genetically similar diploid sporophytes in a warm-water ocean farm. HST was measured as chlorophyll autofluorescence per genotype, presented here as fluorescent intensity values. This correlation suggests a predictive relationship between the growth performance of the early microscopic gametophyte stage HST and the later macroscopic sporophyte stage, indicating the potential for selecting resilient kelp strains under warmer ocean temperatures. However, HST kelps showed reduced genetic variation, underscoring the importance of integrating heat tolerance genes into a broader genetic pool to maintain the adaptability of kelp populations in the face of climate change.

Metagenome-Assembled Genomes from Photo-Oxidized and Nonoxidized Oil-Degrading Marine Microcosms.

We performed deep metagenomic sequencing on hydrocarbon-degrading marine microcosms designed to experimentally determine the effect of photo-oxidation on oil biodegradation dynamics. Assembly, binning, and dereplication yielded 73 unique metagenome-assembled genomes (MAGs) from 6 phyla, of which 61 are predicted to be over 90% complete.

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.

A robust algorithm for explaining unreliable machine learning survival models using the Kolmogorov-Smirnov bounds.

A new robust algorithm based on the explanation method SurvLIME called SurvLIME-KS is proposed for explaining machine learning survival models. The algorithm is developed to ensure robustness to cases of a small amount of training data or outliers of survival data. The first idea behind SurvLIME-KS is to apply the Cox proportional hazards model to approximate the black-box survival model at the local area around a test example due to the linear relationship of covariates in the model. The second idea is to incorporate the well-known Kolmogorov-Smirnov bounds for constructing sets of predicted cumulative hazard functions. As a result, the robust maximin strategy is used, which aims to minimize the average distance between cumulative hazard functions of the explained black-box model and of the approximating Cox model, and to maximize the distance over all cumulative hazard functions in the interval produced by the Kolmogorov-Smirnov bounds. The maximin optimization problem is reduced to the quadratic program. Various numerical experiments with synthetic and real datasets demonstrate the SurvLIME-KS efficiency.

The natural language explanation algorithms for the lung cancer computer-aided diagnosis system.

Two algorithms for explaining decisions of a lung cancer computer-aided diagnosis system are proposed. Their main peculiarity is that they produce explanations of diseases in the form of special sentences via natural language. The algorithms consist of two parts. The first part is a standard local post-hoc explanation model, for example, the well-known LIME, which is used for selecting important features from a special feature representation of the segmented lung suspicious objects. This part is identical for both algorithms. The second part is a model which aims to connect selected important features and to transform them to explanation sentences in natural language. This part is implemented differently for both algorithms. The training phase of the first algorithm uses a special vocabulary of simple phrases which produce sentences and their embeddings. The second algorithm significantly simplifies some parts of the first algorithm and reduces the explanation problem to a set of simple classifiers. The basic idea behind the improvement is to represent every simple phrase from vocabulary as a class of the "sparse" histograms. An implementation of the second algorithm is shown in detail.

A Pipeline for Classifying Deleterious Coding Mutations in Agricultural Plants.

The impact of deleterious variation on both plant fitness and crop productivity is not completely understood and is a hot topic of debates. The deleterious mutations in plants have been solely predicted using sequence conservation methods rather than function-based classifiers due to lack of well-annotated mutational datasets in these organisms. Here, we developed a machine learning classifier based on a dataset of deleterious and neutral mutations in Arabidopsis thaliana by extracting 18 informative features that discriminate deleterious mutations from neutral, including 9 novel features not used in previous studies. We examined linear SVM, Gaussian SVM, and Random Forest classifiers, with the latter performing best. Random Forest classifiers exhibited a markedly higher accuracy than the popular PolyPhen-2 tool in the Arabidopsis dataset. Additionally, we tested whether the Random Forest, trained on the Arabidopsis dataset, accurately predicts deleterious mutations in Orýza sativa and Pisum sativum and observed satisfactory levels of performance accuracy (87% and 93%, respectively) higher than obtained by the PolyPhen-2. Application of Transfer learning in classifiers did not improve their performance. To additionally test the performance of the Random Forest classifier across different angiosperm species, we applied it to annotate deleterious mutations in Cicer arietinum and validated them using population frequency data. Overall, we devised a classifier with the potential to improve the annotation of putative functional mutations in QTL and GWAS hit regions, as well as for the evolutionary analysis of proliferation of deleterious mutations during plant domestication; thus optimizing breeding improvement and development of new cultivars.

Lehr's fields of campaniform sensilla in beetles (Coleoptera): functional morphology. II. Wing reduction and the sensory field.

Loss of the flight ability and wing reduction has been reported for many taxa of Coleoptera. If elytra are closed, their roots are clenched between the tergum and the pleuron, forces applied to the elytra can not be transmitted to the field of campaniform sensilla situated on the root. That is why it is plausible to assume that the field becomes redundant in non-flying beetles. We examined the relationships between the hind wing reduction and characters of this mechanosensory field in beetles of six families. We measured the size of the elytron, that of the hind wing and counted the number of sensilla in the sensory field. Mesopterous non-flying beetles retain one half to one third of sensilla present in macropterous species of the same body size. Further reduction of the sensory field in brachypterous species is obvious, but sensilla are still present in insects with strongly reduced wings, as long as their elytra are separable and mesothoracic axillaries are present. Complete loss of sensilla coincides with the existence of a permanent sutural lock. However, some beetles with permanently locked elytra and absence of axillaries still retain few campaniform sensilla. A very special case of an extreme wing modification in feather-wing beetles is considered. No sensilla were revealed either on the root of the elytron or on the basal segment of such fringed wings in flying ptiliid species.