目的 通过CRISPR/Cas9技术构建尿酸氧化酶（urate oxidase，UOX）对应的Uox基因敲除且能稳定遗传的小鼠品系，并评价其是否能够模拟高尿酸血症（hyperuricemia，HUA）及相关并发症的疾病特点。

方法 首先，使用CRISPR/Cas9基因敲除系统配合显微注射技术构建Uox基因敲除小鼠，通过PCR技术结合DNA测序鉴定首建鼠及子代鼠的基因型和所敲除序列，繁育获得Uox^-/-小鼠，并通过Western Blot检测敲除后Uox基因的蛋白表达情况。之后，采用酶比色法评价Uox^-/-小鼠的尿酸水平，采用血清生化、组织病理、CT扫描等实验评价Uox^-/-小鼠的肾脏、胰腺、肝脏、脑、骨骼的组织损伤水平，通过qPCR、免疫荧光、免疫组化探究肾脏、胰腺以及脑组织中的炎症反应水平。最后通过分析粪便和肾脏代谢组学数据，筛选Uox^-/-小鼠粪便和肾脏中的差异代谢物，分析具有促炎作用的关键介质。

结果 成功敲除Uox基因的Exon 2片段，使得肝脏组织失去了UOX蛋白的表达，构建获得Uox^-/-小鼠，并在Uox^-/-小鼠体内检测到符合HUA临床诊断标准的高水平尿酸。Uox^-/-小鼠出现肾脏功能下降，伴有中重度的肾小管囊性扩张、变性、纤维化和肾小球肥大增生；肝功能下降，伴有中等量肝细胞变性和脂肪堆积；以及高血糖、高脂血症、痛风性关节炎和骨质疏松等症状。并且在肾脏、胰腺以及脑组织中检测到NLRP3炎症小体及IL-1β、IL-18、IL-6的mRNA水平升高和巨噬细胞（或小胶质细胞）浸润。从Uox^-/-小鼠的粪便代谢组和肾脏代谢中，筛选到以糖基化类、吲哚类、酚类为代表的多种尿毒症毒素的含量上调。

结论 通过CRISPR/Cas9技术成功获得了Uox基因Exon 2敲除的小鼠纯合品系，可以作为HUA及相关并发症研究的动物模型，也为相关药物的研发及评价提供了重要工具。

Objective CRISPR/Cas9 is used to construct the uricase-deficient mouse with stable inheritance, which is evaluated whether it can simulate the disease characteristics of hyperuricemia and related complications.

Methods Firstly, uricase deficient mouse model were constructed using CRISPR/Cas9 knockout system combined with microinjection technology. The genotypes and knockout sequences of the first mouse and its offspring were identified by PCR technology combined with DNA sequencing. Uox^-/- mice were obtained by breeding, and the protein expression of Uox gene after knockout was detected by Western blot. After that, the uric acid level of Uox^-/- mice was evaluated by enzyme colorimetry, and the tissue damage levels of kidney, pancreas, liver, brain and bone of Uox^-/-mice were evaluated by biochemical detection, histopathology, CT scanning and other experiments. qPCR, immunofluorescence and immunohistochemistry were used to explore the inflammatory reaction levels in kidney, pancreas and brain tissues. Finally, the metabolomic data of feces and kidneys were analyzed to screen the differential metabolites in the feces and kidneys of Uox^-/- mice and analyze the key mediators with pro-inflammatory effects.

Results The Exon 2 fragment of Uox gene was successfully knocked out, making the liver tissue lose the expression of UOX protein. Uox^-/- mice were successfully constructed, and high levels of uric acid meeting the clinical diagnostic criteria of HUA were detected in Uox^-/- mice. Uox^-/- mice showed decreased renal function, accompanied by moderate to severe renal tubular cystic dilatation, degeneration, fibrosis, and glomerular hypertrophy and hyperplasia; Liver function decreased, accompanied by moderate amounts of hepatocyte degeneration and fat accumulation; Uox^-/- mice also had hyperglycemia, hyperlipidemia, gouty arthritis and osteoporosis. The mRNA levels of NLRP3 inflammasome and IL-1β, IL-18, IL-6 increased and macrophages (or microglia) infiltrated in kidney, pancreas, and brain tissue. From the fecal metabolites and renal metabolites of Uox^-/- mice, the contents of a variety of uremic toxins represented by glycosylated, indole, and phenols were screened to be up-regulated.

Conclusions The homozygous uricase-deficient mouse strain is successfully constructed by CRISPR/Cas9 technology , which can be used as an animal model for research in the field of hyperuricemia and related complications, and also provides an important tool for the development and evaluation of related drugs.