Background: Chronic stress is associated with detrimental effects on physical health, such as chronic restraint stress (CRS), which can damage the intestinal tract. Although tryptophan has many benefits in maintaining intestinal health, the underlying mechanism of its protective effects against stress-induced intestinal injury remains unclear. Methods: In this study, we constructed a CRS model by using a behavioral restraint device in which mice were restrained for 6 h per day over 14 days and investigated the effects, as well as the potential mechanism of a high-tryptophan diet (0.4% tryptophan), on CRS-induced intestinal injury using scanning electron microscopy, 16S rRNA sequencing, and LC-MS. Results: A 0.4% tryptophan diet (fed ad libitum for 24 days) attenuated CRS-induced pathologies, including weight loss, elevated corticosterone, intestinal barrier injury, increased permeability, and epithelial apoptosis. Tryptophan modulated the gut microbiota composition in CRS-induced mice, increasing the abundance of Bacteroidota and decreasing the abundance of Firmicutes, as well as enhancing metabolic function through pathways identified by KEGG analysis. Additionally, tryptophan restored the levels of short-chain fatty acids (SCFAs), including acetic, propionic, isobutyric, butyric, and valeric acids. Correlation analyses showed interactions between tryptophan, intestinal permeability, SCFAs, and gut microbiota. Conclusions: Tryptophan supplementation attenuates CRS-induced intestinal injury by modulating intestinal barrier integrity and gut microbiota homeostasis, and the beneficial effects are largely associated with the SCFA-mediated regulation of intestinal permeability and microbiota-associated energy metabolism.

Currently, a major challenge exists in CRISPR-mediated genome editing research in sheep: the low efficiency of exogenous large DNA fragment targeted integration without drug selection or fluorescence enrichment. This restriction significantly impedes the use of precise genome editing in sheep for agricultural, biological, and biomedical purposes. In this study, we employed the strategy of increasing the local concentration of the homologous repair template at the site of the DNA double-strand break (DSB). We achieved this by fusing the DNA binding domain (BD) of the Gal4 protein (Gal4-BD) to the N-terminal end of the SpCas9 protein using a 32-amino acid (aa) flexible linker. Additionally, we incorporated a 17 bp UAS at the 3′ end of the donor template, which can be specifically recognized and bound by Gal-BD. As a result, we observed a significant improvement in the knock-in efficiency of the exogenous large DNA fragment (2997 bp) in sheep fetal fibroblasts (SFFs), increasing it from 5.30% (8/151) to 16.67% (32/192), providing a comparatively efficient and user-friendly method to promote CRISPR-mediated gene knock-in in sheep.