Molekulârnaâ biologiâ

"Molekulârnaâ biologiâ" covers a wide scope of problems related to molecular, cell and computational biology including genomics, proteomics, bioinformatics, molecular virology and immunology, molecular development biology, and molecular evolution. Molecular Biology publishes reviews, mini-reviews, experimental and theoretical works, short communications. Annualy, the journal publishes special issues devoted to most rapidly developing branches of physical-chemical biology and to the most outstanding scientists on the occasion of their anniversary birthdays. The authors of the journal are from Russia and other countries of the World.
"Molekulârnaâ biologiâ" is indexed/abstracted in Science Citation Index Expanded (SciSearch), Journal Citation Reports/Science Edition, SCOPUS, Chemical Abstracts Service (CAS), Google Scholar, EBSCO Discovery Service, CSA, CAB International, Academic OneFile, Academic Search, AGRICOLA, Biological Abstracts, Biological and Agricultural Index, BIOSIS, CAB Abstracts, CSA Environmental Sciences, EMBiology, Expanded Academic, Global Health, Health Reference Center Academic, Highbeam, INIS Atomindex, OCLC, OmniFile, Science Select, SCImago, Summon by ProQuest, Zoological Record, Microbiology Abstracts Section B: Health & Safety Science Abstracts, Virology and AIDS Abstracts.

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Vol 57, No 3 (2023)

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Improvement of Crops Using the CRISPR/Cas System: New Target Genes
Ukhatova Y.V., Erastenkova M.V., Korshikova E.S., Krylova E.A., Mikhailova A.S., Semilet .V., Tikhonova N.G., Shvachko N.A., Khlestkina E.K.

Successful application of the CRISPR/Cas genome editing system to various crops largely depends on the correct choice of target genes that may be purposefully changed to improve yield, quality, and resistance to biotic and abiotic stressors. The objective of this work was systematizing and cataloguing the information on the confirmed target genes for crop improvement. The latest systematic review was presented on peer-reviewed scientific papers (indexed in the Scopus database) published before August 17, 2019. The present study covers the period from August 18, 2019 to March 15, 2022. The search according to the given algorithm revealed 2090 publications, and their analysis showed that only 685 original papers contained the results of gene editing for 28 crops (the search included 56 crops). A significant part of these publications described the application of genome editing to target genes previously identified in similar works or the studies were associated with reverse genetics, while only 136 publications contained data on editing new target genes whose modification was aimed at improving plant traits important for breeding. The total number of target genes in cultivated plants that were edited to improve properties of breeding value over the entire period of the CRISPR/Cas system application was 287. A detailed analysis of the editing of new target genes is presented in this review. The studies were most often aimed at increasing plant productivity and disease resistance as well as improving the properties of plant materials. Observations are made whether it was possible to obtain stable transformants at the time of publication and whether the editing technique was applied to non-model cultivars. For a number of crops, however, the range of modified cultivars was significantly expanded, specifically for wheat, rice, soybean, tomato, potato, rapeseed, grapevine, and maize. In a vast majority of cases, agrobacterium-mediated transformation was used to deliver the editing construct; less often it was bioballistics, protoplast transfection or haploinducers. The desired change in traits was most often achieved by gene knockout. In some cases, knockdown and nucleotide substitutions were applied. The base-editing and prime-editing approaches have increasingly been used to make nucleotide substitutions in crop genes. The emergence of a convenient CRISPR/Cas editing system helped to significantly intensify the development of molecular genetics specific to many crop species.

Molekulârnaâ biologiâ. 2023;57(3):387-410
pages 387-410 views
Human rDNA Structure, Expression, and Non-Canonical Functions: the Role of Non-Coding Regions
Sadova A.A., Panteleev D.Y., Pavlova G.V.

The review is dedicated to analyzing and summarizing the data on the part of human genome encoding 45S rRNA. The sequences which seem evolutionary conserved on the first glance astonish one with their variability in structure and a variety of functions on closer examination. The major part of rDNA is non-coding and contains regulatory elements, protein binding sites, pseudogenes, repetitive sequences, and microRNA genes. Ribosomal intergenic spacers are not only in charge with the nucleolus morphology and functioning, namely, the rRNA expression and ribosome biogenesis, but also control nuclear chromatin formation thus mediating cell differentiation. Besides, alterations in the expression of these non-coding regions of rDNA in response to environmental stimuli underlies the keen sense of cell to various types of stressors. Malfunctioning of this process may result in a wide range of pathologies from oncology to neurodegenerative disease and mental illness. Here we observe to-date materials on the structure and transcription of the ribosomal intergenic spacer in humans and its role in rRNA expression, in-born disease development, and cancer.

Molekulârnaâ biologiâ. 2023;57(3):411-426
pages 411-426 views
How Histone Deacetylase 3 Controls Hepcidin Expression and Hepatitis C Virus Replication
Shcherbakova А.S., Kochetkov S.N., Kozlov M.V.

The key role of histone deacetylases (HDACs) in the regulation of the cellular response to infection with the hepatitis C virus (HCV) was first demonstrated in 2008. Studying the metabolism of iron in the liver tissues of patients with chronic hepatitis C, the authors found that the expression of the hepcidin gene (HAMP), a hormone regulator of iron export, is markedly reduced in hepatocytes under conditions of oxidative stress caused by viral infection. HDACs were involved in the regulation of hepcidin expression through the control of acetylation level of histones and transcription factors, primarily STAT3, associated with the HAMP promoter. The purpose of this review is to summarize current data on the functioning of the HCV-HDAC3-STAT3-HAMP regulatory circuit as an example of a well-characterized interaction between the virus and the epigenetic apparatus of the host cell.

Molekulârnaâ biologiâ. 2023;57(3):427-439
pages 427-439 views
DNA Fragment Enrichment for High-Throughput Sequencing
Sinyakov A.N., Kostina E.V.

Application of oligonucleotides, mainly obtained using new generation DNA synthesizers (microarray DNA synthesizers), for the enrichment of targeted genomic fragments are described. Methods of molecular hybridization, polymerase chain reaction and CRISPR-based methods for targets enrichment are considered. Examples of the practical use of the developed methods for research and diagnostic purposes are given.

Molekulârnaâ biologiâ. 2023;57(3):440-457
pages 440-457 views


Transcriptional Analysis of the Gene Encoding the Putative Myristoylated Membrane Protein (ORF458R) of Invertebrate Iridescent Virus 6 (IIV6)
Kuz C.A., Ozsahin E., Nalcacioglu R., Demirbag Z.

Invertebrate iridescent virus 6 (IIV6) is a member of the genus Iridovirus and belongs to the Iridoviridae family. The entirely sequenced dsDNA genome, composed of 212 482 bp, encodes 215 putative open reading frames (ORFs). ORF458R encodes a putative myristoylated membrane protein. RT-PCR analysis of ORF458R expression in the presence of DNA replication and protein synthesis inhibitors showed that this gene is transcribed in the late phase of the virus infection. Time course analysis showed that transcription of ORF458R initiates between 12 and 24 h p.i. and starts to decrease after this point. Transcription of ORF458R initiated 53 nucleotides upstream of the translation start site and ended 40 nucleotides after the stop codon. Dual luciferase reporter gene assay showed that sequences between ‒61st and +18th nucleotides are essential for promoter activity. Interestingly, a remarkable decrease in promoter activity, in the presence of sequences between ‒299th and ‒143rd nucleotides, suggested a repressor activity between these regions. Our results showed that ORF458R is transcriptionally active, and separately located sequences at its upstream region with promoter and repressor activities regulating its expression. This information on the transcriptional analysis of ORF458R will contribute to our understanding of the molecular mechanisms of IIV6 replication.

Molekulârnaâ biologiâ. 2023;57(3):458-459
pages 458-459 views
Differential Expression of a Foreign Gene in Arabidopsis Mitochondria
Tarasenko V.I., Tarasenko T.A., Gorbenko I.V., Konstantinov Y.M., Koulintchenko M.V.

Genetic transformation of higher eukaryotes mitochondria in vivo is one of the unresolved and important problems. For efficient expression of foreign genetic material in mitochondria, it is necessary to select regulatory elements that ensure a high level of transcription and transcript stability. This work is aimed at studying the effectiveness of regulatory elements of mitochondrial genes flanking exogenous DNA using the phenomenon of natural competence of plant mitochondria. For this purpose, genetic constructs carrying the GFP gene under the control of the promoter regions of the RRN26 or COX1 genes and one of the two 3'-untranslated regions (3'-UTR) of mitochondrial genes were imported into isolated Arabidopsis mitochondria, followed by transcription in organello. It was shown that the level of GFP expression under the control of promoters of the RRN26 or COX1 genes in organello correlates with the level of transcription of these genes observed in vivo. At the same time, the presence of the tRNAТrp sequence in the 3'-UTR leads to a higher level of the GFP transcript than the presence in this region of the 3'-UTR of the NAD4 gene containing the binding site of the MTSF1 protein. The results obtained open up prospects for creating a system for efficient transformation of the mitochondrial genome.

Molekulârnaâ biologiâ. 2023;57(3):460-470
pages 460-470 views
Identification of Functionally Significant Polymorphic Variants in miRNA Genes in Carotid Atherosclerosis
Zarubin A.A., Mannanova K.V., Koroleva I.A., Sleptсov A.A., Kuznetsov M.S., Kozlov B.N., Nazarenko M.S.

miRNAs are vital molecules of gene expression. They are involved in the pathogenesis of various common diseases, including atherosclerosis, its risk factors and complications. A detailed characterization of the spectrum of functionally significant polymorphisms of miRNA genes of patients with advanced carotid atherosclerosis is an actual research task. We analyzed miRNA expression and exome sequencing data of carotid atherosclerotic plaques of the same male patients (n = 8, 66–71 years of age, 67‒90% degree of carotid artery stenosis). For further study and analysis of the association between rs2910164 polymorphism of the MIR146A gene and advanced carotid atherosclerosis, we recruited 112 patients and 72 relatively healthy Slavic residents of Western Siberia. 321 and 97 single nucleotide variants (SNVs) were detected in the nucleotide sequences of pre- and mature miRNAs in carotid atherosclerotic plaques. These variants were located in 206 and 76 miRNA genes, respectively. Integration the data of exome sequencing and miRNA expression revealed 24 SNVs of 18 miRNA genes which were processed to mature form in carotid atherosclerotic plaques. SNVs with the greatest potential functional significance for miRNA expression predicted in silico were rs2910164:C>G (MIR146A), rs2682818:A>C (MIR618), rs3746444:A>G (MIR499A), rs776722712:C>T (MIR186), rs199822597:G>A (MIR363). The expression of miR-618 was lower in carotid atherosclerotic plaques of patients with the AC rs2682818 genotype of the MIR618 gene compared with the CC genotype (log2FC = 4.8; p = 0.012). We also found the association of rs2910164:C (MIR146A) with the risk of advanced carotid atherosclerosis (OR = 2.35; 95% CI: 1.43–3.85; p = 0.001). Integrative analysis of polymorphism in miRNA genes and miRNA expression is informative for identifying functionally significant polymorphisms in miRNA genes. The rs2682818:A>C (MIR618) is a candidate for regulating miRNA expression in carotid atherosclerotic plaques. The rs2910164:C (MIR146A) is associated with the risk of advanced carotid atherosclerosis.

Molekulârnaâ biologiâ. 2023;57(3):471-482
pages 471-482 views
The Profile of microRNA Expression in Diffuse Large B-Cell Lymphoma
Veryaskina Y.A., Titov S.E., Kovynev I.B., Fyodorova S.S., Shebunyaeva Y.Y., Antonenko O.V., Pospelova T.I., Zhimulev I.F.

Non-Hodgkin’s lymphoma (NHL) is a heterogeneous group of cancers characterized by different pathogenesis and prognosis. The main methods for treating NHL are chemotherapy, immunochemotherapy, and radiation therapy; however, most of these cancers are known to be chemoresistant or return rapidly after the short chemotherapy-induced remission. Therefore, searching for alternative cytoreductive therapy options is quite relevant today. Aberrant microRNA (miRNA) expression is one of the mechanisms responsible for the emergence and progression of lymphoid malignancies. This study was aimed at identifying the miRNA expression profile in diagnostic biopsy specimens harvested from the lymph nodes affected by diffuse large B-cell lymphoma (DLBCL) and identifying miRNA markers, which can potentially be used to design a novel type of ta-rgeted anticancer drugs that would allow one to achieve maximum therapy personalization and increase its efficacy. The key study objects were histological specimens harvested from the lymph nodes by excisional d-iagnostic biopsy and treated using the conventional histomorphological formalin fixation methods. The study group consisted of patients with DLBCL (n = 52). The biopsy specimens harvested from patients with reactive lymphadenopathy (RL) (n = 40) constituted the control group. The miR-150 expression level was reduced over 12-fold (p = 3.6 × 10‒15) compared to that in the tissues of non-cancerous nodular masses. B-ioinformatic analysis revealed that miR-150 is involved in regulation of hematopoiesis and lymphopoiesis. The findings obtained in this study allow considering miR-150 a promising therapeutic target having a great potential for clinical applications.

Molekulârnaâ biologiâ. 2023;57(3):492-500
pages 492-500 views


Involvement of the Gene, a Domesticated Gene of Retrovirus, in the Stress Response Pathway in Different Species
Gigin A.N., Nefedova L.N.

The Gagr gene is a domesticated gag retroelement gene in Drosophila melanogaster, whose function is associated with a stress response. The protein products of the Gagr gene and its homologues in different Drosophila species have a highly conserved structure; however, they demonstrate a certain variability in the promoter region of the gene, apparently associated with the gradual acquisition of a new function and involvement in new signaling pathways. In this work we studied the effect of oxidative stress caused by ammonium persulfate on the survival of various species of the genus Drosophila (D. melanogaster, D. mauritiana, D. simulans, D. yakuba, D. teissieri, D. pseudoobscura), analyzed the correlation between the structure of promoter regions and stress-induced changes in the expression of the Gagr gene and its homologues in different Drosophila species and comparison of stress-induced changes in the expression of oxidative stress markers: Jak-STAT signaling pathway activator gene upd3, Jak-STAT pathway effector vir-1, and signaling pathway target IMD Rel. It was found that in D. simulans and D. mauritiana sensitivity to ammonium persulfate is significantly increased, which correlates with a reduced level of transcription of vir-1 gene orthologues. The latter is due to a decrease in the number of binding sites for the transcription factor STAT92E, a component of the Jak-STAT signaling pathway, in the vir-1 promoter region. Consistent changes in the expression of the Gagr, upd3, vir-1 genes are observed in all species of the melanogaster subgroup, except D. pseudoobscura, which indicates an increase in the role of Gagr in the regulation of stress response pathways during the phylogenesis of the genus Drosophila.

Molekulârnaâ biologiâ. 2023;57(3):483-491
pages 483-491 views


Effects of Peroxisome Proliferator-Activated Receptor γ on Modulating Angiopoietin-Like Protein 4 Synthesis in Caco-2 Cells Exposed to
Zhao X., Huang H.S., Shi S.R.

Angiopoietin-like protein 4 (ANGPTL4) is considered to be one of the important circulating mediators linking intestinal microorganisms and host lipid metabolism. The objective of this study was to assess the effects of peroxisome proliferator-activated receptor γ (PPARγ) on modulating ANGPTL4 synthesis in Caco-2 cells exposed to Clostridium butyricum. The viability of Caco-2 cells and the expression of PPARγ and ANGPTL4 in Caco-2 cells were detected after the Caco-2 cells were co-cultured with C. butyricum at the concentration of 1 × 106, 1 × 107 and 1 × 108 CFU/mL. The results showed that cell viability was enhanced by C. butyricum. Besides, PPARγ and ANGPTL4 expression and secretion in Caco-2 cells was significantly increased by 1 × 107 and 1 × 108 CFU/mL of C. butyricum. Furthermore, the effects of PPARγ on modulating ANGPTL4 synthesis in Caco-2 cells regulated by 1 × 108 CFU/mL of C. butyricum was also be expounded in PPARγ activation/inhibition model based on Caco-2 cells and via ChIP technique. It was found that C. butyricum promoted the binding of PPARγ to the PPAR binding site (chr19: 8362157-8362357, located upstream of the transcriptional start site of angptl4) of the angptl4 gene in Caco-2 cells. However, the PPARγ was not the only way for C. butyricum to stimulate ANGPTL4 production. Taken together, PPARγ played a role in the regulation of ANGPTL4 synthesis by C. butyricum in Caco-2 cells.

Molekulârnaâ biologiâ. 2023;57(3):501-502
pages 501-502 views
EBF1 Promotes the Sensitivity of Cervical Cancer Cells to Cisplatin via Activating FBN1 Transcription
Shen N.N., Lin J.H., Liu P.P.

Cisplatin (DDP) is widely used in the chemotherapy of cervical cancer (CC), the fourth most common female malignancy worldwide. However, some patients progress to chemotherapy resistance, which leads to chemotherapy failure, tumor recurrence, and poor prognosis. Therefore, strategies to identify the regulatory mechanisms underlying CC development and increase tumor sensitivity to DDP will help improve patient survival. This research was designed to ascertain the mechanism of EBF1-dependent regulation of FBN1 which promotes chemosensitivity of CC cells. The expression of EBF1 and FBN1 was measured in CC tissues resistant or sensitive to chemotherapy and in DDP-sensitive or -resistant cells (SiHa and SiHa-DDP cells). SiHa-DDP cells were transduced with lentiviruses encoding EBF1 or FBN1 to evaluate the influence of these two proteins on cell viability, expression of MDR1 and MRP1, and cell aggressiveness. Moreover, the interaction between EBF1 and FBN1 was predicted and demonstrated. Finally, to further verify the EBF1/FB1-dependent mechanism of DDP sensitivity regulation in CC cells a xenograft mouse model of CC was established using SiHa-DDP cells transduced with lentiviruses carrying EBF1 gene and shRNA directed to FBN1.EBF1 and FBN1 showed decreased expression in CC tissues and cells, particularly in those resistant to chemotherapy. Transduction of SiHa-DDP cells with lentiviruses encoding EBF1 or FBN1 lead to decreased viability, IC50, proliferation capacity, colony formation ability, aggressiveness, and increased cell apoptosis. We have shown that EBF1 activates FBN1 transcription by binding to FBN1 promoter region. Additionally, it was revealed that FBN1 silencing reversed the promoting effect of EBF1 overexpression on chemosensitivity of CC cells in vivo. EBF1 facilitated chemosensitivity in CC cells by activating FBN1 transcription.

Molekulârnaâ biologiâ. 2023;57(3):503-504
pages 503-504 views
Development of a Series of Neutralizing Nanobodies against SARS-CoV-2 Spike Protein
Zhuchkov V.A., Ivanov S.V., Kravchenko J.E., Chumakov S.P.

Countering the spread of new respiratory infections and reducing the damage they cause to society requires efficient strategies for rapid development of targeted therapeutics, such as monoclonal antibodies. Nanobodies, defined as variable fragments of heavy-chain camelid antibodies, have a set of characteristics that make them particularly convenient for this purpose. The speed at which the SARS-CoV-2 pandemic had spread has confirmed that a key factor in the development of therapeutics is obtaining highly effective blocking agents as soon as possible, as well as the diversity of epitopes to which these agents bind. We have optimized the process of selection of blocking nanobodies from the genetic material of camelids and obtained a panel of nanobody structures with affinity to spike protein in the lower nanomolar and picomolar ranges and high binding specificity. The subset of nanobodies that demonstrate the ability to block the interaction between the spike protein and the cellular ACE2 receptor was selected in experiments in vitro and in vivo. It has been established that the epitopes bound by the nanobodies are located in the RBD domain of the spike protein and have little overlap. The diversity of binding regions may allow the mixture of nanobodies to retain potential therapeutic efficacy towards new variants of the spike protein, and the structural features of nanobodies, in particular, their compact size and high stability, indicate the possibility of their utilization in the form of aerosols.

Molekulârnaâ biologiâ. 2023;57(3):505-516
pages 505-516 views


DNA Sequence-Specific Ligands. XIX. Synthesis, Spectral Properties, Virological and Biochemical Studies of Fluorescent Dimeric Trisbenzimidazoles DB()
Arutyunyan A.F., Kostyukov A.A., Korolev S.P., Gottikh M.B., Susova .Y., Kaluzhny D.N., Zhuze A.L.

In this work, we synthesized and characterized the properties of a series of new fluorescent narrow-groove ligands DB3(n). DB3(n) compounds based on dimeric trisbenzimidazoles have the ability to bind to the AT regions of DNA. The synthesis of DB3(n), trisbenzimidazole fragments of which are linked by oligomethylene linkers of different lengths (n = 1, 5, 9), is based on the condensation of monomeric trisbenzimidazole MB3 with α,ω-alkyldicarboxylic acids. DB3(n) proved to be effective inhibitors of the catalytic activity of HIV-1 integrase at submicromolar concentrations (0.20–0.30 µM). DB3(n) was found to inhibit the catalytic activity of DNA topoisomerase I at low micromolar concentrations.

Molekulârnaâ biologiâ. 2023;57(3):517-527
pages 517-527 views
Oxidative Probing of the G4 DNA Structure by ZnP1 Porphyrin within Sequences of  and Promotors
Chashchina G.V., Kaluzhny D.N.

The formation of G4 structures in a DNA double helix competes with the complementary strand, which can change the equilibrium G4 structures studied on single-strand models by classical structural methods. A relevant task is to develop methods for detecting and localizing G4 in extended double-stranded (ds) DNA in the promoter regions of the genome. The porphyrin derivative ZnP1 selectively binds and leads to photo-induced oxidation of guanine in G4 structures on single-stranded (ss) and dsDNA model systems. In this research, we show the oxidative effect of ZnP1 on native sequences of MYC and TERT oncogene promoters that potentially capable to form G4 structures. Single strand breaks in the guanine rich sequence caused by ZnP1 oxidation and subsequent cleavage of the DNA strand by Fpg glycosylase were identified and assigned to the nucleotide sequence. The detected break sites corresponded to sequences potentially capable of forming G4 structures. New data were obtained on the possibility of folding G4 structures in the presence of a complementary strand in the context of the DNA double helix of the natural sequence.

Molekulârnaâ biologiâ. 2023;57(3):528-536
pages 528-536 views


Exploring the Prognostic Features of Hepatocellular Carcinoma via Text Mining and Data Analysis
Yang Z.H., Wang S.X.

Transcatheter arterial chemoembolization is one of the interventional treatments for hepatocellular carcinoma (HCC). This treatment is generally used for patients with intermediate to advanced hepatocellular carcinoma, and identifying the role of HCC-related genes can help improve the efficiency of transcatheter arterial chemoembolization. To investigate the role of HCC-related genes and to provide valid evidence for transcatheter arterial chemoembolization treatment, we performed a comprehensive bioinformatics analysis. Through text mining (“hepatocellular carcinoma”) and microarray data analysis (GSE104580), we obtained a standard gene set, which was followed by gene ontology and Kyoto Gene and Genome Encyclopedia analysis. The significant 8 genes clustered in protein-protein interactions network were chosen to be used in the follow-up analysis. Through survival analysis low expression of the key genes were found to be strongly associated with survival in HCC patients in this study. The correlation between the expression of the key genes and tumor immune infiltration was assessed by Pearson correlation analysis. As a result, 15 drugs targeting seven of the eight genes have been identified, and therefore can be considered as potential components for transcatheter arterial chemoembolization treatment of HCC.

Molekulârnaâ biologiâ. 2023;57(3):537-538
pages 537-538 views


Recombinase Polymerase Amplification for Rapid Detection of Human Bacterial Pneumonia Pathogens
Lapa S.A., Surzhikov S.A., Blagodatskikh S.A., Shershov V.E., Chudinov A.V.

A diagnostic system based on recombinase polymerase amplification (RPA) has been developed to identify six bacterial pathogens of human pneumonia. Species-specific primers have been designed and optimized to conduct a multiplex reaction in one common volume. Labeled primers were used for reliable discrimination of amplification products close in size. Identification of the pathogen was carried out by visual analysis of the electrophoregram. The analytical sensitivity of the developed multiplex RPA was 102‒103 copies of DNA. The specificity of the system was determined by the absence of cross-amplification of the studied DNA samples of pneumonia pathogens for each pair of primers, as well as for the DNA of Mycobacterium tuberculosis H37, and amounted to 100%. The analysis execution time is less than an 1 h, including electrophoretic reaction control. The test system can be used in specialized clinical laboratories for rapid analysis of samples from patients with suspected pneumonia.

Molekulârnaâ biologiâ. 2023;57(3):539-545
pages 539-545 views
Type II CRISPR-Cas System Nucleases: a Pipeline for Prediction and Characterization
Vasileva А.A., Aliukas S.A., Selkova P.A., Arseniev A.N., Chernova V.E., Musharova O.S., Klimuk E.I., Khodorkovskii M.A., Severinov K.V.

The use of CRISPR-Cas bacterial adaptive immunity systems components for targeted DNA changing has opened broad prospects for programmable genome editing of higher organisms. The most widely used gene editor-s are based on the Cas9 effectors of the type II CRISPR-Cas systems. In complex with guide RNAs, Cas9 proteins are able to directionally introduce double-strand breaks into DNA regions complementary to guide RNA sequences. Despite the wide range of characterized Cas9s, the search for new Cas9 variants remains an actual task, since the available Cas9 editors have several limitations. This paper presents a workflow for the search and subsequent characterization of new Cas9 nucleases developed in our laboratory. Detailed protocols describing the bioinformatical search, cloning and isolation of recombinant Cas9 proteins, testing for nuclease activity in vitro, and determining the PAM sequence required for recognition of DNA targets, are presented. Potential difficulties that may arise, as well as ways to overcome them, are considered.

Molekulârnaâ biologiâ. 2023;57(3):546-560
pages 546-560 views

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