The Greater Caucasus is a part of seismically active Alpine–Himalayan orogenic belt and has been a center of significant volcanic activity during the Quaternary period. That led to the formation of the number of hydrothermal habitats, including subterranean thermal aquifers and surface hot springs. However, there are only a limited number of scientific works reporting on the microbial communities of these habitats. Moreover, all these reports concern only studies of specific microbial taxa, carried out using classical cultivation approaches. In this work, we present first culture-independent study of hydrotherms in the Republic of North Ossetia-Alania, located in the southern part of the North Caucasus. Using 16S metabarcoding, we analyzed the composition of the microbial communities of two subterranean thermal aquifers and terrestrial hot springs of the Karmadon valley. Analysis of correlations between the chemical composition of water and the representation of key taxa allowed us to identify the key factors determining the formation of microbial communities. In addition, we were able to identify a significant number of highly abundant deep phylogenetic lineages. Our study represents a first glance on the thermophilic microbial communities of the North Caucasus and may serve as a basis for further microbiological studies of the extreme habitats of this region.

Genome editing technologies that are currently available and described have a fundamental impact on the development of molecular biology and medicine, industrial and agricultural biotechnology and other fields. However, genome editing based on detection and manipulation of the targeted RNA is a promising alternative to control the gene expression at the spatiotemporal transcriptomic level without complete elimination. The innovative CRISPR-Cas RNA-targeting systems changed the conception of biosensing systems and also allowed the RNA effectors to be used in various applications; for example, genomic editing, effective virus diagnostic tools, biomarkers, transcription regulations. In this review, we discussed the current state-of-the-art of specific CRISPR-Cas systems known to bind and cleave RNA substrates and summarized potential applications of the versatile RNA-targeting systems.

Type V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from Ruminococcus bromii sp. entitled RbCas12a, which is able to efficiently cleave target DNA templates, using the particularly high accessibility of PAM 5′-YYN and a relatively wide temperature range from 20 °C to 42 °C. In comparison to Acidaminococcus sp. (AsCas12a) nuclease, RbCas12a is capable of processing DNA more efficiently, and can be active upon being charged by spacer-only RNA at lower concentrations in vitro. We show that the human-optimized RbCas12a nuclease is also active in mammalian cells, and can be applied for efficient deletion incorporation into the human genome. Given the advantageous properties of RbCas12a, this enzyme shows potential for clinical and biotechnological applications within the field of genome editing.

The development of RNA-targeting CRISPR-Cas systems represents a major step forward in the field of gene editing and regulation. RNA editing presents a viable alternative to genome editing in certain scenarios as it offers a reversible and manageable approach, reducing the likelihood of runaway mutant variants. One of the most promising applications is in the treatment of genetic disorders caused by mutations in RNA molecules. In this study, we investigate a previously undescribed Cas12g nuclease which was found in metagenomes from promising thermophilic microbial communities during the expedition to the Republic of North Ossetia—Alania in 2020. The method outlined in this study can be applied to other Cas orthologs and variants, leading to a better understanding of the CRISPR-Cas system and its enzymatic activities. The cis-cleavage activity of the new type V-G Cas effector was indicated by in vitro RNA cleavage experiments. While CRISPR-Cas systems are known for their high specificity, there is still a risk of unintended cleavage of nontargeted RNA molecules. Ultimately, the search for new genome editing tools and the study of their properties will remove barriers to research in this area. With continued research and development, we may be able to unlock their full potential.

The phylum Armatimonadota represents one of the least characterized bacterial lineages, with only three formally described species despite its widespread distribution in various environments. Deep subsurface thermal environments harbor significant microbial diversity and represent promising sources for novel enzyme discovery through metagenomic approaches. This study reports the identification, cloning, and biochemical characterization of Cel7465, a novel endoglucanase from an uncultured GBS-DC family within the order Fimbriimonadales. The enzyme was identified through metagenomic analysis of microbial mats from the Biragzang deep thermal well (North Ossetia, Russia, 48 °C) and successfully expressed in cells of Escherichia coli strain ArcticExpress (DE3). Phylogenetic analysis assigned Cel7465 to glycoside hydrolase family 5 subfamily 36. The purified recombinant enzyme exhibited optimal activity at 55 °C and pH 8.0, with high specific activity of 4347 U/mg. The enzyme demonstrated broad pH tolerance (50% activity retained from pH 4.0 to 10.0) and notable thermal stability, retaining 60% activity after one hour at 80 °C and 20% after four hours. Remarkably, the presence of Mn²⁺ ions enhanced enzyme activity more than 7-fold, while Mg²⁺ and Ca²⁺ ions provided moderate activation. Cel7465 represents the first biochemically characterized glycoside hydrolase from the order Fimbriimonadales, expanding our understanding of enzymatic capabilities within the understudied phylum Armatimonadota and demonstrating the continued potential of extreme environments as sources of novel industrial biocatalysts.