The use of CRISPR/Cas nucleases for the development of DNA diagnostic systems in out-of-laboratory conditions (point-of-need testing, PONT) has demonstrated rapid growth in the last few years, starting with the appearance in 2017-2018 of the first diagnostic platforms known as DETECTR and SHERLOCK. The platforms are based on a combination of methods of nucleic acid isothermal amplification with selective CRISPR/Cas detection of target amplicons. This significantly improves the sensitivity and specificity of PONT, making them comparable with or even superior to the sensitivity and specificity of polymerase chain reaction, considered as the "gold standard" of DNA diagnostics. The review considers modern approaches to the coupling of CRISPR/Cas detection using Cas9, Cas12a, Cas12b, Cas13a, Cas14, and Cas3 nucleases to various methods of nucleic acid isothermal amplification, with an emphasis on works in which sensitivity at the level of single molecules (attomolar and subattomolar concentrations of the target) is achieved. The properties of CRISPR/Cas nucleases used for targeted DNA diagnostics and the features of methods of nucleic acid isothermal amplification are briefly considered in the context of the development of diagnostic biosensing platforms. Special attention is paid to the most promising directions for the development of DNA diagnostics using CRISPR/Cas nuclease.

Genitourinary cancer (GUC) represents more than one fifth of all human cancers. This makes the development of approaches to its early diagnosis an important task of modern biomedicine. Circulating microRNAs, short (17-25 nucleotides) non-coding RNA molecules found in human biological fluids and performing a regulatory role in the cell, are considered as promising diagnostic and prognostic biomarkers of cancers, including GUC. In this review we have considered the current state of research aimed at assessing microRNAs as biomarkers of such human GUC types as malignant tumors of the bladder, kidney, prostate, testicles, ovaries, and cervix. A special attention has been paid to studies devoted to the identification of microRNAs in urine as a surrogate "liquid biopsy" that may provide the simplest and cheapest approach to mass non-invasive screening of human GUC. The use of microRNA panels instead of single types of microRNA generally leads to higher sensitivity and specificity of the developed diagnostic tests. However, to date, work on the microRNAs assessment as biomarkers of human GUC is still of a research nature, and the further introduction of diagnostic tests based on microRNAs into practice requires successful clinical trials.

Using the method of shotgun mass spectrometry, we have evaluated changes in the proteomic profile of HaCat cells in response to the treatment with sodium dodecyl sulfate (anionic surfactant) and Triton-X100 (non-ionic surfactant) in two concentrations (12.5 µg/ml and 25.0 µg/ml). The study revealed induction of orphan CYP2S1 (biotransformation phase I) in response to Triton-X100. We have identified proteins of II (glutathione-S-transferases, GSTs) and III (solute carrier proteins, SLCs) biotransformation phases, as well as antioxidant proteins (peroxiredoxins, PRDXs; catalase, CAT; thioredoxin, TXN). Thus, proteins of all three xenobiotic detoxification phases were detected. The presented results suggest a new prospect of using HaCaT keratinocytes as a model of human epidermis for studying the metabolism of drugs/toxicants in human skin in vitro.

Recombinase polymerase and loop-mediated isothermal amplification can be conducted in nonlaboratory conditions, making these methods promising for the development of rapid tests for the DNA diagnosis of infectious diseases in humans, agricultural animals, and plants in the “point-of-care” testing or “in-field” detection format. The review discusses the fundamental principles underlying these methods and describes their current status, with a focus on noninstrumental methods for the detection of results in isothermal amplification using colorimetry and lateral flow assays. Approaches to enhancing the selectivity of isothermal amplification through its combination with CRISPR/Cas detection or by combining two methods based on the “nested amplification” principle are thoroughly examined.

Using analytical technologies it is possible now to measure the entire diversity of molecules even in a small amount of biological samples. Metabolomic technologies simultaneously analyze thousands of low-molecular substances in a single drop of blood. Such analytical performance opens new possibilities for clinical laboratory diagnostics, still relying on the measurement of only a limited number of clinically significant substances. However, there are objective difficulties hampering introduction of metabolomics into clinical practice. The Institute of Biomedical Chemistry (IBMC), consolidating the efforts of leading scientific and medical organizations, has achieved success in this area by developing a clinical blood metabogram (CBM). CBM opens opportunities to obtain overview on the state of the body with the detailed individual metabolic characteristics of the patient. A number of scientific studies have shown that the CBM is an effective tool for monitoring the state of the body, and based on the CBM patterns (signatures), it is possible to diagnose and monitor the treatment of many diseases. Today, the CBM creation determines the current state and prospects of clinical metabolomics in Russia. This article, dedicated to the 80th anniversary of IBMC, is a review of these achievements focused on a discussion of their implementation in clinical practice.

The work demonstrates that recombinant CRISPR-nuclease Cas12a, purified after heterologous expression with a simplified method using single-stage metal-chelate chromatography, can be successfully utilized in DETECTR technology. The combination of CRISPR-nuclease Cas12a obtained in such a way with recombinase polymerase amplification (RPA) allowed one to ensure the selectivity of detection of Dickeya solani—the dangerous bacterial phytopathogen causing the potato disease known as “blackleg”—against closely related and unrelated bacterial phytopathogens with a limit of detection of one copy of the bacterial genome per amplification reaction. The result can be determined visually, without the use of complex instrumental methods, by changing the color of the reaction sample when illuminated with blue light that creates the basis for development of field DNA diagnostics of D. solani. The use of simplified chromatographic purification will significantly reduce the time and resources required to obtain a functionally active CRISPR-nuclease Cas12a for development and production of DNA diagnostics based on DETECTR technology.

The approach to characterize preparations of recombinant Cas13a-nuclease in terms of specific collateral activity has been proposed for standardization of enzyme preparations. The standardization of Cas13a preparations by the specific activity may benefit both the development of assays employing Cas13a collateral ribonuclease activity and the optimization of ribonuclease expression, purification, and storage. The approach is based on measurement of the initial rate of a cleavage of specially designed commercially available RNA molecules (�reporters� labelled with a fluorophore and a quencher) by a preparation of recombinant Cas13a-nuclease and commercial RNase A of known activity. This requires the optimization of a molar ratio for the formation of Cas13a complexes with guide RNA as well as the optimization of amount of the RNA target. The use of a synthetic RNA target appears preferable compared with total RNA preparations.

Using human chromosome 18 (Ch18) genes as an example, a PCR analysis of the interindividual variability of expression of 244 genes was performed in the liver tissue. Although the quantitative profiles of the Ch18 transcriptome, expressed as the number of cDNA copies per single cell, showed a high degree of correlation between donors (Pearson correlation coefficient r(p) ranged from 0.963 to 0.966), the expression of the significant number of genes (from 13% to 19%, depending on the method of experimental data normalization) varied by more than 4-fold when comparing donors pairwise. At the same time, the proportion of differentially expressed genes increased with a decrease in the level of their expression. It is shown that the higher quantitative variability of low-abundance transcripts has mainly biological rather than technical nature. Bioinformatic analysis of the interindividual variability of the differential expression of Ch18 genes in the human liver tissue did not reveal any statistically significant groups of genes related to certain biological processes. This obviously indicates a rather transient nature of the interindividual variability of Ch18 gene expression, probably reflecting the response of cells of an individual to specific external stimuli.

Direct electrochemical determination of double-stranded DNA fragments obtained as a result of polymerase chain or recombinase polymerase reactions, using deoxyuridine triphosphates modified by the aromatic group of tyrosine as a substrate, was used to detect the causative agent of potato bacteriosis Dickeya solani.

The analysis of cytochrome P450 transcripts was carried out by the nanopore sequencing in liver tissue samples of three donors and HepG2 line cells. It has been demonstrated that direct mRNA sequencing with a MinION nanopore sequencer (Oxford Nanopore Technologies) allows one to obtained quantitative profiles for transcripts (and their splice variants) of cytochrome P450 superfamily genes encoding isoforms involved in metabolism of the large (~80%) part of drugs. The splice variant profiles substantially differ for donors. The cytochrome P450 gene expression at the transcript level is significantly weaker in cells of the HepG2 line compared with that in the normal liver tissue. This limits the capability of the direct mRNA nanopore sequencing for studying alternative splicing of cytochrome P450 transcripts in HepG2 cells. Both quantitative and qualitative profiles of the cytochrome P450 gene expression at the transcript level are notably differ in human liver tissue and HepG2 cells.

The paper describes an original technique for detecting the collateral activity of CRISPR/Cas 13a ribonuclease based on the assessment of ribosomal RNA degradation. The Agilent 2100 bioanalyzer is used as an analyzing device. This approach is an alternative to existing detection methods and has a number of advantages over them in the case when a quantitative assessment of activity is not required. On the example of the test sample, the optimal concentrations and ratios of the components of the reaction mixture, which are necessary to obtain the most indicative result, were determined. The proposed technique can be used for qualitative assessment of the activity of recombinant ribonuclease Cas13a preparations obtained under different conditions of heterologous protein expression and purification, as well as for testing guide RNAs.

The proteomic composition of a biological sample serves as the most important feature of a biological object, and it allows discriminating normal and pathological conditions. Targeted mass spectrometric analysis, namely, multiple reaction monitoring (MRM) using synthetic isotopically-labeled internal standard (SIS), is the main alternative to the ELISA method for the analysis of diagnostically significant proteins. Based on the MRM results, a prototype test system has been developed; it employs the targeted mass spectrometric method for multiplex, quantitative analysis of FDA-verified proteins in whole blood plasma. Using this approach, it was possible to measure the content of 42 proteins in 31 samples in a concentration range spanning five orders of magnitude. The interindividual variability for 30 of the 42 registered proteins was less than 40%. The largest scatter was observed for haptoglobin (68%), immunoglobulin heavy constant delta IGHD (90%), angiotensin (72%), sex hormone-binding globulin SHBG (100%) and lipoprotein-(a) (136%). The obtained results on the concentration of proteins correlate with published data (Hortin et al., 2008, Clinical Chemistry, 54, 1608) with R2=0.84. The developed prototype test system based on targeted mass spectrometric analysis of proteins can be considered as an alternative to methods using monoclonal antibodies.

Studies of genomes and transcriptomes are performed using sequencers that read the sequence of nucleotide residues of genomic DNA, RNA, or complementary DNA (cDNA). The analysis consists of an experimental part (obtaining primary data) and bioinformatic processing of primary data. The bioinformatics part is performed with different sets of input parameters. The selection of the optimal values of the parameters, as a rule, requires significant computing power. The article describes a protocol for processing transcriptome data by virtual computers provided by the cloud platform Amazon Web Services (AWS) using the example of the recently emerging technology of long DNA and RNA sequences (Oxford Nanopore Technology). As a result, a virtual machine and instructions for its use have been developed, thus allowing a wide range of molecular biologists to independently process the results obtained using the "Oxford nanopore".

An effect of primer strand slippage was observed in the primer extension reaction due to replacement of thymidine triphosphate for modified analogs of dUTP. The effect was detected when using Vent (exo–) DNA polymerase. It is notably suppressed by adding to reaction mixture the natural nucleotides dATP, dCTP, and dGTP.

In the frame of the work, data on the implementation of metabolomics tests in medicine have been systematized. Based on the obtained data, a set of protocols was proposed, the sequential realization of which makes it possible to conduct a blood metabolome analysis for medical purposes. Using this analysis and the number of blood samples from healthy volunteers, a prototype of a healthy person's metabolomic image has been developed; it allows visually and digitally to assess the compliance of the human blood metabolome with the norm. At the same time, 99% of the metabolic processes reflected in the blood plasma are estimated. If abnormalities are detected, the metabolomic image allows to get the value of these deviations of metabolic processes in digital terms.

A method of comparative analysis of scientific trajectories based on bibliographic profiles has been proposed. The bibliographic profile ("meshprint") is a list of MeSH terms (key terms used to index articles in the PubMed), indicating the relative frequency of occurrence of each term in the scientist's articles. Comparison of personalized bibliographic profiles can be represented in the form of a semantic network, where the nodes are the names of scientists, and the relationships are proportional to the calculated measures of similarity of bibliographic profiles. The proposed method was used to analyze the semantic network of scientists united by the academic school of academician A.I. Archakov. The results of this work demonstrate the relationship between the scientific trajectories of one scientific school and to correlate the results with world trends.

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).