Hypoxanthine-guanine phosphoribosyltransferase1 (HPRT1)基因突变会导致次黄嘌呤和鸟嘌呤代谢异常,严重的代谢障碍被称为Lesch-Nyhan综合征,表现为智力低下,行为上出现强迫性自我毁伤,并伴随痛风或肾结石等症状。HPRT1基因具有多种突变模式,近年来对其突变谱的研究表明该基因具有一些“突变热点”。本研究选取了导致翻译提前终止的热点突变c.508C>T突变和c.151C>T突变,在HEK293T和HeLa细胞中,以单链寡核苷酸(single-stranded oligo-deoxyribonucleotides, ssODN)作为同源重组模板,利用CRISPR/Cas9技术构建了这两种突变的单克隆细胞株,发生定点突变的效率分别为16.3%和10%。进一步通过Western blot检测点突变的单克隆细胞的HPRT1蛋白表达水平,通过6-TG毒性实验检测点突变的单克隆细胞的次黄嘌呤/鸟嘌呤磷酸核糖转移酶活性,结果表明纯合突变的单细胞克隆不能正常表达HPRT1蛋白,其次黄嘌呤/鸟嘌呤磷酸核糖转移酶活性丧失;杂合突变的单细胞克隆的HPRT1蛋白表达量降低,仍具有部分次黄嘌呤/鸟嘌呤磷酸核糖转移酶活性。HPRT1基因定点突变细胞模型的建立可为今后建立其他HPRT1基因突变的细胞系或动物模型提供借鉴,为研究Lesch-Nyhan致病机制提供基础。

Mutations in Hypoxanthine-guanine Phosphoribosyltransferase1 (HPRT1) gene can lead to metabolic disorder of hypoxanthine and guanine metabolism, and other severe symptoms such as hypophrenia, gout, and kidney stones, called the Lesch-Nyhan disease (LND). Although the mutations are widely distributed throughout the HPRT1 gene, there are some isolated hot spots. In this study, we aim to introduce two previously reported hot spots, c.508 C>T and c.151 C>T, which could lead to premature translational termination in HPRT1 gene. Through CRISPR/Cas9 mediated homology-directed repair (HDR) by using single-stranded oligo-deoxyribonucleotides (ssODN) as donor template, we obtained cell clones containing these two mutations in HEK293T or HeLa cells. Targeted mutation of c.508 C>T and c.151 C>T reached to 16.3% and 10%, respectively. We further detect HPRT1 protein levels with Western blot and enzyme activity with 6-TG in 5 different cell clones. HPRT1 protein and its enzymatic activity both was hardly detected in homozygous mutant cells, while reduced HPRT1 protein expression and enzymatic activity was detected in heterozygous mutant cells. Our study will be beneficial to those who working on generation of cell or animal models of HRPT1 mutations, and provides a basis for further investigations on the genetic mechanism of Lesch-Nyhan disease.