Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA.

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5'-scaffold moiety and variable 3'-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.

Mitochondrial Protein SLIRP Affects Biosynthesis of Cytochrome c Oxidase Subunits in HEK293T Cells.

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.

Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from Ruminococcus bromii.

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.

Beyond the Surface: A Deeper Look at the Psychosocial Impacts of Acne Scarring.

Post-acne scarring is a common consequence of acne vulgaris with no universal cure. Although there have been many recent advances to address acne scars physically, there is still a lack of research that investigates their psychosocial impacts. Our comprehensive PubMed search presents an overview of existing information to highlight known sources of mental distress caused by post-acne scarring, both related to and independent of the psychosocial detriments caused early on by active acne. The literature indicates that acne scarring is a distinct condition from acne vulgaris and therefore requires a comprehensive clinical approach unique from those available for active acne.

Subcision for Atrophic Acne Scarring: A Comprehensive Review of Surgical Instruments and Combinatorial Treatments.

Subcutaneous incisionless surgery, also known as subcision, is a minimally invasive procedure that is commonly indicated for the treatment of atrophic acne scars. In recent years, many new techniques have been developed to maximize results from this procedure. This review article aims to identify an updated list of instruments and combinatorial treatments available for atrophic acne scar patients undergoing subcision. We constructed a comprehensive PubMed search term and performed triple-blinded screening on all resulting studies for mentions of subcision as indicated by acne scarring. Our results show that there are four main categories of subcision tools that are commonly employed to treat atrophic acne scars: needles, cannulas, wires, and blunt-blade instruments. Usage of these devices varies by scar depth, personal preference, and combinatorial treatment options. Overall, subcision is a particularly effective treatment for atrophic acne scars, and there is vast potential for further innovation with this technique.

A Comprehensive Review of Non-Energy-Based Treatments for Atrophic Acne Scarring.

Scarring is a dire consequence of acne vulgaris. Particularly, atrophic acne scarring is highly prevalent among young adults, and its physical and psychological effects can persist throughout their lives if left untreated. This literature review will analyze various non-energy-based approaches to treating atrophic acne scarring, emphasizing recent advances within the last 5 to 10 years. To accomplish this, we performed a PubMed search for various acne scar treatments such as chemical peels, dermabrasion, microdermabrasion, subcision, microneedling, punch techniques, dermal fillers, and thread lifting. Our findings and analysis show that there is no panacean solution to treating atrophic acne scars, which explains the evolving trend towards developing unique combinatorial treatments. Although a fair comparison of each treatment approach is difficult to achieve due to the studies' varying sample sizes, strength of evidence, treatment execution, etc, there still remains a level of consensus on what treatments are well suited for particular scar types.