上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (1): 8-15.doi: 10.3969/j.issn.1674-8115.2021.01.002

• 基础研究 • 上一篇    下一篇

CRISPR/Cas9介导的同源重组在非编码区疾病相关位点功能研究中的应用

徐宁(), 周甜, 侯国俊, 沈南, 唐元家()   

  1. 上海交通大学医学院附属仁济医院风湿病科,上海市风湿病学研究所,上海 200127
  • 出版日期:2021-01-28 发布日期:2021-02-22
  • 通讯作者: 唐元家 E-mail:sjtuxuning@foxmail.com;yjtang@sibs.ac.cn
  • 作者简介:徐宁(1993—),男,硕士生;电子信箱:sjtuxuning@foxmail.com
  • 基金资助:
    国家自然科学基金(81871287);上海交通大学医学院高水平地方高校创新团队(SSMU-ZDCX20180100)

Application of CRISPR/Cas9-mediated homologous recombination to the functional study of disease-associated genetic variants located in non-coding region

Ning XU(), Tian ZHOU, Guo-jun HOU, Nan SHEN, Yuan-jia TANG()   

  1. Department of Rheumatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Institute of Rheumatology, Shanghai 200127, China
  • Online:2021-01-28 Published:2021-02-22
  • Contact: Yuan-jia TANG E-mail:sjtuxuning@foxmail.com;yjtang@sibs.ac.cn
  • Supported by:
    Funding Information] National Natural Science Foundation of China(81871287);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20180100)

摘要:

目的·以全基因组关联研究报道的系统性红斑狼疮相关单核苷酸多态性(single nucleotide polymorphism,SNP)位点 rs1978421为例,利用规律成簇间隔短回文重复序列/相关蛋白9[clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9,CRISPR/Cas9]系统建立非编码区疾病相关的遗传突变位点功能的研究策略。方法·针对SNP rs1978421位点设计单链导向RNA(single guide RNA,sgRNA),利用T7核酸内切酶Ⅰ(T7 endonuclease Ⅰ,T7EⅠ)实验在HEK293T细胞中验证sgRNA对靶点的切割效率。以电穿孔转染的方式将融合Cas9-绿色荧光蛋白(green fluorescent protein,GFP)和sgRNA的表达质粒及同源重组的模板转染至U-937细胞内,通过流式分选得到单个的GFP阳性U-937细胞,培养14 d后进行基因型鉴定。筛选得到同源重组的细胞克隆后,通过实时荧光定量PCR(quantitative real-time PCR,qPCR)对rs1978421上下游2 Mbp范围内的基因及miR-146a进行定量,分析rs1978421不同等位基因对miR-146a及其周围基因的表达影响。结果·T7EⅠ实验表明,约有24%的HEK293T细胞中的rs1978421位点发生切割。在U-937细胞内进行的同源重组实验中,共计得到141个细胞株;其中6个同源重组成功,成功率为4.3%。qPCR结果显示,在野生型TT与同源重组成功CC的U-937细胞中,miR-146a及所检测基因的表达情况均无显著变化(均P>0.05)。结论·SNP rs1978421对其周围2 Mb范围内的基因及miR-146a并无调控作用,但该研究证明了CRISPR介导的同源重组技术用于非编码区SNP位点功能研究的可行性。

关键词: 规律成簇间隔短回文重复序列, 同源重组, 全基因组关联研究, 单核苷酸多态性

Abstract:

Objective·To take the systemic lupus erythematosus (SLE)-associated single nucleotide polymorphism (SNP) rs1978421 reported in the genome-wide association study (GWAS) as an example, and establish the research strategy for the functional study of disease-associated genetic variants located in the non-coding region through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system.

Methods·Single guide RNA (sgRNA) was designed for SNP rs1978421. T7 endonuclease Ⅰ (T7E Ⅰ) experiment was used to verify the cleavage efficiency of sgRNA in HEK293T cells. The homologous recombinant template and expression plasmid of Cas9-green fluorescent protein (GFP)/sgRNA were transfected into U-937 cells by electroporation transfection method. GFP positive U-937 single cells were sorted by flow cytometry, and the cells were cultured for the following 14 days. Then the genotype identification was carried out. After the homologous recombinant cell clones were screened, quantitative real-time PCR (qPCR) was used to quantify the upstream and downstream genes of rs1978421 within 2 Mb and miR-146a, and the effects of different alleles of rs1978421 on the expression of miR-146a and its surrounding genes were analyzed.

Results·T7E Ⅰ assay showed that about 24% of the total rs1978421 sites in HEK293T were cleaved. In homologous recombination experiment of the U-937 cells, a total of 141 cell lines were obtained and only six were successful, while the ratio of homologous recombination was 4.3%. qPCR showed that there was no significant difference in the expression of miR-146a and the detected genes between wild type TT and homologous recombinant CC in the U-937 cells (all P>0.05).

Conclusion·SNP rs1978421 has no regulatory effect on the surrounding genes within 2 Mb and miR-146a. However, this study proves the feasibility of CRISPR-mediated homologous recombination technology for the functional study of SNP sites in the non-coding regions.

Key words: clustered regularly interspaced short palindromic repeats (CRISPR), homologous recombination, genome-wide association study (GWAS), single nucleotide polymorphism (SNP)

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