Lesch-Nyhan综合征(Lesch-Nyhan syndrome，LNS)是一种先天性的嘌呤代谢缺陷病，次黄嘌呤鸟嘌呤磷酸核糖转移酶(hypoxanthine phosphoribosyltransferase，HPRT)是其主要致病基因，临床特征表现为尿酸分泌过多、痛风、肾结石及肾脏损伤等，目前致病机制尚未被完全阐明且无有效治愈手段。动物模型在疾病致病机理研究和治疗方式探索中发挥着重要功能。本研究采用高效的CRISPR/Cas9基因编辑技术和显微注射的方式敲除家兔(Oryctolagus cuniculus)HPRT基因建立了LNS家兔模型，以期能更好的模拟该疾病的表型。首先针对兔HPRT基因第3外显子设计一条sgRNA，将体外转录的sgRNA和Cas9 mRNA共注射到兔受精卵中，注射后的胚胎移植到代孕母兔子宫中，待仔兔出生后对其基因型及表型进行鉴定与分析。共出生4只仔兔(分别编号为R1、R2、R3和R4)，测序结果显示4只仔兔都产生了不同程度的基因修饰，基因编辑效率达100%，其中R4仔兔T载体测序结果在基因编辑靶点未检测到野生型序列。通过6-硫代鸟嘌呤(6-Thioguanine，6-TG)药物测试，证明R4仔兔HPRT酶活性缺失；肾组织HE染色发现4只仔兔表现出肾小球炎细胞浸润、集合管脱落等肾脏损失。本研究成功设计了1条能够敲除家兔HPRT基因的sgRNA，并采用CRISPR/Cas9基因编辑技术和显微注射方式成功构建了HPRT基因修饰家兔，为研究LNS综合征致病机制和治疗方式提供了新的非啮齿类动物模型。

Lesch-Nyhan syndrome (LNS) is a congenital defect disease that results in defective purine metabolism. It is caused by pathogenic variants of the HPRT gene. Its clinical symptoms mainly include high uric acid levels, gout, and kidney stones and damage. The mechanism of LNS has not been fully elucidated, and no cure exists. Animal models have always played an important role in exploring causative mechanisms and new therapies. This study combined CRISPR/Cas9 and microinjection to knock out the HPRT gene to create an LNS rabbit model. A sgRNA targeting exon 3 of HPRT gene was designed. Subsequently, Cas9 mRNA and sgRNA were injected into rabbit zygotes, and injected embryos were transferred to the uterus. The genotype and phenotype of rabbits were analyzed after birth. Four infant rabbits (named R1, R2, R3 and R4), which showed varying levels of gene modification, were born. The gene-editing efficiency was 100%. No wild-type sequences at the target HPRT gene were detected in R4 rabbit. Next, 6-thioguanine drug testing confirmed that HPRT enzymatic activity was deficient in R4 infant rabbit. HE staining revealed kidney abnormalities in all infant rabbits. Overall, an sgRNA capable of knocking out the HPRT gene in rabbits was successfully designed, and HPRT gene-modified rabbits were successfully constructed by using CRISPR/Cas9 technology and microinjection. This study provides a new nonrodent animal model for studying LNS syndrome.