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

doi:10.3969/j.issn.1674-8115.2021.01.002

摘要/Abstract

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)

中图分类号:

Q3-3

徐宁, 周甜, 侯国俊, 沈南, 唐元家. 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[J]. 上海交通大学学报(医学版), 2021, 41(1): 8-15.

Ning XU, Tian ZHOU, Guo-jun HOU, Nan SHEN, Yuan-jia TANG. Application of CRISPR/Cas9-mediated homologous recombination to the functional study of disease-associated genetic variants located in non-coding region[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(1): 8-15.

图/表 8

表1 rs1978421位点鉴定引物

Tab 1 Indentification primer of rs1978421 locus

Primer	Sequence (5'→3')
rs1978421-F	TACTTTTTGGCATCCCTGCT
rs1978421-R	GACCAACCTCGGCAACATAG

表2 sgRNA功能序列及单链引物

Tab 2 sgRNA functional sequence and single strand primers

Name	Sequence (5'→3')
sgRNA functional sequence	TTTCATTCCATCACCAAGTC
sgRNA-F	caccgTCTCATTCCATCACCAAGTC
sgRNA-R	aaacGACTTGGTGATGGAATGAGAc
U6 promoter sequencing primer	TGGACTATCATATGCTTACC

表3 同源重组模板序列

Tab 3 Sequence of homologous recombination template

Template	Sequence (5'→3')
rs1978421HR	CCAAACCTGGGCAACAACCCATAAATACATGTAGCCTT-TTTTTTTTTTCTGAGACAGAGTCTCATTCCATCACCAA-GTCTGGAGTGCAGTGGTGCGATCATGGCTCACTGCAG-CCTGGACC

表4 qPCR引物序列

Tab 4 qPCR primers

Primer	Sequence (5'→3')
GAPDH-F	GGAGCGAGATCCCTCCAAAAT
GAPDH-R	GGCTGTTGTCATACTTCTCATGG
PTTG1-F	ACCCGTGTGGTTGCTAAGG
PTTG1-R	ACGTGGTGTTGAAACTTGAGAT
SLU7-F	AAGAACAGCGAAAATTGGGCA
SLU7-R	CCCTCTTGTACCATTCTCCAGA
TTC1-F	GAGCGGACAAGGTTGAGAACA
TTC1-R	CTCCTCCTTTAGTCTAGTGCTCT
PWWP2A-F	CTTGTCGTGTCGTTCCGCTT
PWWP2A-R	ACCATTTGCTTCACACTTGACTT
UBLCP1-F	CTCGCAGAGTGAAAGAGTACAAA
UBLCP1-R	GCACAAGACCTGTGGTCAAATA

图1 非编码区SNP位点研究策略及rs1978421位点sgRNA设计及切割效率检测Note:A. Schematic diagram of the research strategy for SNP located in non-coding region. B. Wild type U-937 SNP rs1978421 genotype identification (reverse primer-mediated sequencing). C. pSpCas9(BB)-2A-GFP plasmid element instructions (NLS—nuclear location signal). D. T7EⅠ experiments for sgRNA cutting efficiency test in HEK293T cells (M—marker; FI—fluorescence intensity).

Fig 1 Research strategy for SNP located in non-coding region and rs1978421 sgRNA design and cutting efficiency test

图2 流式分选与细胞基因型鉴定Note:A. FACS analyzed the GFP positive U-937 cell ratio. B. Genotypes of part of homozygous cell clones. C. Sequencing data of rs1978421 in successfully recombined clones and wild type clones.

Fig 2 FACS sorting and cell clones genotype identification

表5 SNP rs1978421同源重组实验所得基因型细胞株汇总

Tab 5 Summary of genotypes of cell lines obtained in SNP rs1978421 homologous recombination

Genotype	Count/n	Ratio/%
Heterozygote	86	61.0
Wild type	25	17.7
Successfully recombinated (T-C)	6	4.3
Insert (1 bp)	5	3.5
Insert (65 bp)	4	2.8
Deletion (2 bp)	9	6.4
Deletion (8 bp)	6	4.3

图3 不同基因型细胞中rs1978421邻近基因表达分析Note:A. Neighbor genes within 2 Mbp around rs1978421 [IL12B—Homo sapiens interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40); ADRA1B—Homo sapiens adrenoceptor alpha 1B; FABP6—Homo sapiens fatty acid binding protein 6, ileal; CCNJL—Homo sapiens cyclin J-like; C1QTNF2—Homo sapiens C1q and tumor necrosis factor related protein 2; miR3142HG—transcript miR3142HG; miR3142—Homo sapiens microRNA 3142; ZBED8—Homo sapiens zinc finger BED-type containing 8; ATP10B—Homo sapiens ATPase, class Ⅴ, type 10B; GABRB2—Homo sapiens gamma-aminobutyric acid (GABA) A receptor, beta 2; GABRA6—Homo sapiens gamma-aminobutyric acid (GABA) A receptor, alpha 6]. B. miR-146a expressions in wild type, homologous recombined rs1978421 cell lines and rs1978421 downstream DNA deletion or insertion cell lines （①P=0.898, ②P=0.741, ③P=0.955, compared with TT genotype）. C. rs1978421 neighbor genes expressions in wild type, homologous recombined rs1978421 cell lines and rs1978421 downstream DNA deletion or insertion cell lines （④P=0.408, ⑤P=0.423, ⑥P=0.548, ⑦P=0.543, ⑧P=0.443, compared with TT genotype）.

Fig 3 rs1978421 neighbor genes expressions analysis in cell clones with different genotypes

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