CRISPR-Cas9-mediated uATG introduction in the 5'UTR of the Uox gene for hyperuricemia mouse models: implications for gout and metabolic disorders.

Abstract

Sequence-specific gene knockdown technologies are crucial for fundamental research and therapeutic applications. RNA interference and CRISPR interference, while extensively utilized for gene expression manipulation, face limitations due to their ectopic or transient expression. In this study, we developed a generalizable and efficient method to downregulate gene expression in human 293T cells by introducing de novo upstream ATGs (uATGs) of genes using CRISPR-Cas9-mediated genome editing. Through CRISPR library screening, in-depth sequencing, and flow cytometry analysis, we validated that the introduction of uATGs served as an effective method to suppress protein expression. Our findings further revealed that this strategy can be tailored to diminish endogenous gene expression in tumor cells without affecting the mRNA transcription levels. Importantly, by introducing a uATG into the 5' untranslated region (UTR) of the Uox gene, we successfully established a Uox-knockdown (KD) mouse model of hyperuricemia associated with metabolic disorders. This model demonstrated hyperuricemia, with serum uric acid levels that exceeded 400 µmol L, along with renal dysfunction, as indicated by elevated serum creatinine and blood urea nitrogen levels. Examination of the kidneys from 8-week-old Uox-KD mice revealed abnormal histopathological characteristics, including partial dilation of Bowman's capsules and renal tubules, focal nephron collapse and necrosis, and lymphocytic infiltration. In addition, the mice exhibited lipid and glucose metabolism disorders, all while maintaining a normal lifespan. This spontaneous hyperuricemia model has potential as a valuable tool for long-term studies on hyperuricemia and gout. Taken together, we present an efficient approach for the constant suppression of specific gene expression in mammalian cells and the development of a Uox-KD mouse model of hyperuricemia via CRISPR-Cas9-mediated uATG introduction. This offers broad implications for fundamental research and therapeutic applications.