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.

This review is devoted to synthetic biology. In this field, molecular biology methods and techniques are applied to develop a predetermined cell behavior b

Mitochondria carry out various vital roles in eukaryotic cells, including ATP energy synthesis, the regulation of apoptosis, Fe-S cluster formation, and the metabolism of fatty acids, amino acids, and nucleotides. Throughout evolution, mitochondria lost most of their ancestor’s genome but kept the replication, transcription, and translation machinery. Protein biosynthesis in mitochondria is specialized in the production of highly hydrophobic proteins encoded by mitochondria. These proteins are components of oxidative phosphorylation chain complexes. The coordination of protein synthesis must be precise to ensure the correct assembly of nuclear-encoded subunits for these complexes. However, the regulatory mechanisms of mitochondrial translation in human cells are not yet fully understood. In this study, we examined the contribution of the SLIRP protein in regulating protein biosynthesis in mitochondria. Using a click-chemistry approach, we discovered that deletion of the SLIRP gene disturbs mitochondrial translation, leading to the dysfunction of complexes I and IV, but it has no significant effect on complexes III and V. We have shown that this protein interacts only with the small subunit of the mitochondrial ribosome, which may indicate its involvement in the regulation of the mitochondrial translation initiation stage.

Ribosomes are macromolecular machines of conveyor type, which move along the mRNA from triplet to triplet and polymerize cognate amino acids. They are regarded as uniform entities with constant molecular composition bearing no regulatory capacity. However, their ability to interact with multiple proteins involved in translation suggests existence of specialized ribosomes dedicated to biosynthesis of particular proteins. This can be easily imagined in yeast mitochondria, whose genomes encode eight polypeptides, and where protein-specific translation is already represented by translational activators – a group of proteins each regulating a single mRNA translation. Despite this, the subject remains poorly investigated. We report an exploratory approach to search for distinct populations of ribosomes in the yeast mitochondria. Affinity tags were added to mitochondrial ribosomal proteins, ribosomes were isolated by immunoprecipitation and their protein and RNA content was characterized. It was shown that the mitochondrial ribosomes isolated from yeast strains bearing affinity tags on different ribosomal proteins recover different sets of components. The differences were rather quantitative than qualitative, because almost full sets of mitochondrial ribosomal proteins were identified in each sample, but the ratios demonstrated variance ranging from 10 to less than 0.05 molecules per ribosome. In addition, we explored the power of translational activators as a bait to recover ribosomes translating specific mRNAs in yeast mitochondria.