Enhancing prime editor flexibility with coiled-coil heterodimers.

BACKGROUND: Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency. RESULTS: Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol. CONCLUSIONS: Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.

Acupuncture at Back-Shu and Front-Mu Acupoints Prevents Gastric Ulcer by Regulating the TLR4/MyD88/NF-κB Signaling Pathway.

PURPOSE: To assess the preventive effects of acupuncture at back-shu and front-mu acupoints on rats with restraint water-immersion stress (RWIS)-induced gastric ulcer. METHODS: Thirty-six rats were randomly divided into four groups for 10 days of treatment as follows: the normal group received no treatment; the model group received RWIS-induced gastric ulcer; the omeprazole group was administered omeprazole orally every 2 days; and the electroacupuncture group received electroacupuncture at the RN12 and BL21 acupoints every 2 days. After 10 days of treatment, except for the normal group, all rats were induced with gastric ulcer by RWIS for 3 h. The ulcer index (UI), ulcer inhibition rate, and histopathological score were calculated. We determined the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in serum, and the activities of myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), nitric oxide (NO), and glutathione peroxidase (GSH-Px) in serum and gastric tissues. Protein expression of MyD88, nuclear factor (NF)-κB (p65), and toll-like receptor (TLR) 4 was quantified in gastric tissues. RESULTS: The electroacupuncture and omeprazole groups were equivalent in terms of UI, ulcer inhibition rate, and histopathological score. The serum levels of TNF-α and IL-6 were significantly lower in the electroacupuncture group compared with the omeprazole group (P < 0.05). Compared with the model group, there were significant changes in the levels of NO, MPO, GSH-Px, and MDA in all other groups, while the expression of TLR4, MyD88, and NF-κB p65 in gastric tissue decreased significantly in the electroacupuncture group. The expression of TLR4 was substantially lower in the electroacupuncture group compared with the omeprazole group. CONCLUSION: Acupuncture at back-shu and front-mu acupoints played a role in preventing gastric ulcer by inhibiting extracellular signals, stimulating kinases in serum and gastric tissues, and activating the inhibition of the TLR4 signaling pathway.

Protective effects of genipin on ethanol-induced acute gastric injury in mice by inhibiting NLRP3 inflammasome activation.

Genipin has been shown to exert anti-inflammatory effects, but its mechanism in protecting the ethanol-induced acute gastric injuries remains largely unclear. The present study aimed to investigate the effects of genipin on ethanol-induced acute gastric injuries in mice. After intragastrical administration of genipin for 7 consecutive days, acute gastric injuries were induced in the mice by ethanol treatment for 1 h. The expression levels of MDA, MPO, SOD, CAT, and NO in gastric tissues, and the levels of IL-6, TNF-α, MTL, SP, VIP and SS in serum samples were measured by ELISA. In addition, Western blotting was used to determine the expression levels of proteins involved in NLRP3 signaling pathway. The findings revealed that oral administration of genipin significantly ameliorated the pathological injury of gastric mucosa induced by ethanol, decreased the oxidative stress induced by ethanol and suppressed the expression levels of in-flammatory cytokines in gastric tissues and serum samples. In addition, it was observed that oral administration of genipin could remarkably inhibit the expression levels of related proteins in the NLRP3 signaling pathway. In conclusion, these results suggest that genipin may exhibit protective roles in ethanol-induced gastric mucosal injuries by activating antioxidant system and attenuating inflammatory reaction.