The Development, Optimization and Future of Prime Editing.

Prime editing is a rapidly developing method of CRISPR/Cas-based genome editing. The increasing number of novel PE applications and improved versions demands constant analysis and evaluation. The present review covers the mechanism of prime editing, the optimization of the method and the possible next step in the evolution of CRISPR/Cas9-associated genome editing. The basic components of a prime editing system are a prime editor fusion protein, consisting of nickase and reverse transcriptase, and prime editing guide RNA, consisting of a protospacer, scaffold, primer binding site and reverse transcription template. Some prime editing systems include other parts, such as additional RNA molecules. All of these components were optimized to achieve better efficiency for different target organisms and/or compactization for viral delivery. Insights into prime editing mechanisms allowed us to increase the efficiency by recruiting mismatch repair inhibitors. However, the next step in prime editing evolution requires the incorporation of new mechanisms. Prime editors combined with integrases allow us to combine the precision of prime editing with the target insertion of large, several-kilobase-long DNA fragments.

Studies on glycogen storage disease type 1a animal models: a brief perspective.

Glycogen storage disease type 1 (GSD1) is a rare hereditary monogenic disease characterized by the disturbed glucose metabolism. The most widespread variant of GSD1 is GSD1a, which is a deficiency of glucose-6-phosphatase-ɑ. Glucose-6-phosphatase-ɑ is expressed only in liver, kidney, and intestine, and these organs are primarily affected by its deficiency, and long-term complications of GSD1a include hepatic tumors and chronic liver disease. This article is a brief overview of existing animal models for GSD1a, from the first mouse model of 1996 to modern CRISPR/Cas9-generated ones. First whole-body murine models demonstrated exact metabolic symptoms of GSD1a, but the animals did not survive weaning. The protocol for glucose treatment allowed prolonged survival of affected animals, but long-term complications, such as hepatic tumorigenesis, could not be investigated. Next, organ-specific knockout models were developed, and most of the metabolic research was performed on liver glucose-6-phosphate-deficient mice. Naturally occuring mutation was also discovered in dogs. All these models are widely used to study GSD1a from metabolic and physiological standpoints and to develop possible treatments involving gene therapy. Research performed using these models helped elucidate the role of glycogen and lipid accumulation, hypoxia, mitochondrial dysfunction, and autophagy impairment in long-term complications of GSD1a, including hepatic tumorigenesis. Recently, gene replacement therapy and genome editing were tested on described models, and some of the developed approaches have reached clinical trials.

Acute recovery from disuse atrophy: the role of stretch-activated ion channels in the activation of anabolic signaling in skeletal muscle.

The aim of the study was to 1) measure time-course alternations in the rate of protein synthesis (PS) and phosphorylation status of the key anabolic markers, and 2) find out the role of stretch-activated ion channels (SACs) in the activation of anabolic signaling in the rat soleus during an acute reloading following disuse atrophy. Wistar rats were subjected to 14-day hindlimb suspension (HS) followed by 6, 12, and 24 h of reloading. To examine the role of SAC in the reloading-induced activation of anabolic signaling, the rats were treated with gadolinium (Gd3+), a SAC blocker. The content of signaling proteins was determined by Western blot. c-Myc mRNA expression was assessed by RT-PCR. After 24-h reloading, the PS rate was elevated by 44% versus control. After 6-h reloading, the p-70-kDa ribosomal protein S6 kinase (p70S6k) and translation initiation factor 4E-binding protein 1 (4E-BP1) did not differ from control; however, 12-h reloading resulted in an upregulation of both p70s6k and 4E-BP1 phosphorylation versus control. The phosphorylation of AKT (Ser473) and glycogen synthase kinase-3ß (Ser9) was reduced after HS and then completely restored by 12-h reloading. c-Myc was significantly upregulated during the entire reloading. Gd3+ treatment during reloading (12 h) prevented a full phosphorylation of p70S6k, rpS6, 4E-BP1, as well as PS activation. The results of the study suggest that 1) enhanced PS during the acute recovery from HS may be associated with the activation of ribosome biogenesis as well as mammalian target of rapamycin complex 1 (mTORC1)-dependent signaling pathways, and 2) functional SACs are necessary for complete activation of mTORC1 signaling in rat soleus during acute recovery from HS.