具备高效CRISPR协同激活活性的HIEC6-dCas9-SAM稳转细胞株构建

doi:10.13560/j.cnki.biotech.bull.1985.2024-1033

生物技术通报 ›› 2025, Vol. 41 ›› Issue (5): 52-61.doi: 10.13560/j.cnki.biotech.bull.1985.2024-1033

具备高效CRISPR协同激活活性的HIEC6-dCas9-SAM稳转细胞株构建

任柱平(), 杨泰然, 雷元三, 金留飞, 崔古贞, 田益明(), 陈峥宏()

^1.贵州医科大学基础医学院微生物学教研室，贵阳 550000
^2.贵州省病原生物学特色重点实验室，贵阳 550000
^3.贵州省微生物组与感染性疾病防控重点实验室，贵阳 550000

收稿日期:2024-10-22 出版日期:2025-05-26 发布日期:2025-06-05
通讯作者: 田益明，男，博士，副教授，研究方向：医学微生物学；E-mail: tianym@gmc.edu.cn
陈峥宏，女，博士，教授，研究方向：医学微生物学；E-mail: chenzhenghong@gmc.edu.cn
作者简介:任柱平，男，硕士研究生，研究方向：医学微生物学；E-mail: rzp15685902672@163.com
基金资助:
基金项目:贵州省科技计划项目（黔科合基础-ZK［2023］一般333），贵州医科大学引进博士启动基金项目(校博合J字［2022］010);贵州省病原生物学重点实验室(QJJ［2022］019);高等学校学科创新引智计划(D20009)

Construction of HIEC6-dCas9-SAM Transgenic Cell Line with Highly-efficient CRISPR Synergistic Activation Properties

REN Zhu-ping(), YANG Tai-ran, LEI Yuan-san, JIN Liu-fei, CUI Gu-zhen, TIAN Yi-ming(), CHEN Zheng-hong()

^1.Department of Microbiology, School of Basic Medicine, Guizhou Medical University, Guiyang 550000
^2.Pathogenic Biology Characteristic Laboratory of Guizhou Province, Guiyang 550000
^3.Microbiome and Infectious Diseases Prevention and Control Laboratory of Guizhou Province, Guiyang 550000

Received:2024-10-22 Published:2025-05-26 Online:2025-06-05

摘要/Abstract

摘要：

目的以人正常肠道上皮细胞（HIEC6）为研究模型，建立具有高水平转录激活活性的HIEC6-dCas9-SAM单克隆细胞株，为利用CRISPR激活（CRISPR activation, CRISPRa）系统筛选人类肠道疾病发生发展相关关键基因和探究分子致病机制提供细胞工具。方法首先利用PiggyBac转座子系统构建HIEC6-dCas9-SAM多克隆细胞；然后通过有限稀释法筛选单克隆细胞株，并使用免疫印迹、间接免疫荧光法鉴定单克隆细胞株中dCas9-SAM蛋白（dCas9、VP64、MS2、HSF1、p65）的表达情况；最后利用CRISPRa荧光报告系统和构建包装特定靶基因sgRNA慢病毒，检测所构建稳转株在转录和蛋白水平的CRISPR激活效率。结果成功获得两株HIEC6-dCas9-SAM单克隆细胞，两株细胞均能高水平稳定表达dCas9-SAM蛋白。CRISPRa荧光报告系统检测显示，两株HIEC6-dCas9-SAM稳转细胞的激活效率分别高达96.7%、99.0%。靶基因激活功能验证显示，在转录水平，两株HIEC6-dCas9-SAM稳转细胞中靶基因APN的转录激活水平分别高达2 725倍和4 521倍，SLC35A1基因的转录激活水平分别为27.5倍和18.1倍；在蛋白水平，APN蛋白的激活效率分别高于12.9倍和11.2倍，SLC35A1蛋白的激活效率分别为1.32倍和0.97倍。两株单克隆稳转细胞均表现出较高的转录激活活性。结论成功构建两株具有高水平CRISPR转录激活活性的HIEC6-dCas9-SAM单克隆稳转细胞，为后续基于CRISPRa系统筛选人类肠道疾病发生发展相关关键基因和探究分子致病机制提供了重要细胞工具。

关键词: dCas9-SAM, HIEC6细胞, CRISPR激活, 稳转细胞株

Abstract:

Objective This study aims to establish HIEC6-dCas9-SAM monoclonal cell lines with high-level transcriptional activating activity using human normal intestinal epithelial cells (HIEC6) as a model, which will serve as valuable tools for utilizing the CRISPR activation (CRISPRa) system to screen key genes involved in the pathogenesis of human intestinal diseases and to explore the underlying molecular mechanisms. Method HIEC6-dCas9-SAM polyclonal cells were first constructed using the PiggyBac transposon system. Monoclonal cell lines were then selected using the limited dilution method, and the expressions of dCas9-SAM proteins (dCas9, VP64, MS2, HSF1, and p65) in these monoclonal cell lines was confirmed by Western blotting and indirect immunofluorescence. Finally, CRISPRa fluorescence reporting system and lentivirus-based sgRNA constructs targeting specific genes were employed to evaluate the CRISPRa efficiency of the monoclonal cell lines at both the transcriptional and protein levels. Result Two HIEC6-dCas9-SAM monoclonal cell lines were successfully generated, stably expressing high levels of dCas9-SAM proteins. The CRISPRa fluorescence reporting system demonstrated activation efficiencies of 96.7% and 99.0% for the two cell lines, respectively. Verification of CRISPRa for the target genes revealed that: The transcriptional activation levels of the APN gene in the two HIEC6-dCas9-SAM monoclonal cell lines were as high as 2 725-fold and 4 521-fold respectively, while those of the SLC35A1 gene were 27.5-fold and 18.1-fold, respectively. At the protein level, the activation efficiencies of APN protein were 12.9-fold and 11.2-fold, respectively, while those of SLC35A1 protein were 1.32-fold and 0.97-fold, respectively. Both monoclonal stable cell lines showed high transcriptional activation activity. Conclusion Two HIEC6-dCas9-SAM monoclonal cell lines with highly-efficient CRISPR synergistic activation properties have been successfully established. These cell lines serve as valuable tools for the subsequent screening of key genes involved in the pathogenesis of human intestinal diseases and for exploring the molecular mechanisms underlying these diseases using the CRISPRa system.

Key words: dCas9-SAM, HIEC6 cells, CRISPR activation, stable cell line

任柱平, 杨泰然, 雷元三, 金留飞, 崔古贞, 田益明, 陈峥宏. 具备高效CRISPR协同激活活性的HIEC6-dCas9-SAM稳转细胞株构建[J]. 生物技术通报, 2025, 41(5): 52-61.

REN Zhu-ping, YANG Tai-ran, LEI Yuan-san, JIN Liu-fei, CUI Gu-zhen, TIAN Yi-ming, CHEN Zheng-hong. Construction of HIEC6-dCas9-SAM Transgenic Cell Line with Highly-efficient CRISPR Synergistic Activation Properties[J]. Biotechnology Bulletin, 2025, 41(5): 52-61.

图/表 6

表1 序列信息

Table 1 Sequences information

引物 Primer	序列 Sequence （5′-3′）	注释 Note
sgAPN-F	GAGGAGCCCTGGTGCATCCCGTTTAAGAGCTAGGCCAACATGAGG	sgAPN慢病毒载体引物
sgAPN-R	GGGATGCACCAGGGCTCCTCGGTGTTTCGTCCTTTCCACAAG	sgAPN慢病毒载体引物
sgSLC35A1-F	GGAGGACTACGATTCCCAGAGTTTAAGAGCTAGGCCAACATGAGG	sgSLC35A1慢病毒载体引物
sgSLC35A1-R	TCTGGGAATCGTAGTCCTCCGGTGTTTCGTCCTTTCCACAAG	sgSLC35A1慢病毒载体引物
APN-F	TTCAACATCACGCTTATCCACC	qPCR引物
APN-R	AGTCGAACTCACTGACAATGAAG	qPCR引物
SLC35A1-F	CAACCACAGCCGTGTGTATCA	qPCR引物
SLC35A1-R	TGCTAAGAGCTAGGAAAGCCAT	qPCR引物
Actin-F	CATGTACGTTGCTATCCAGGC	qPCR引物
Actin-R	CTCCTTAATGTCACGCACGAT	qPCR引物

图1 Western blot鉴定阳性多克隆细胞中dCas9-SAM蛋白表达A：dCas9-SAM蛋白结构模拟图（青色：dCas9；红色：VP64；黄色：MS2；棕色：p65；紫色：HSF1）；B：Western blot鉴定阳性细胞dCas9-SAM蛋白表达（1：HIEC6细胞对照；2-14：HIEC6-dCas9-SAM阳性细胞）

Fig. 1 Identification of dCas9-SAM protein expression in HIEC6 positive polyclonal cells via Western blotA: Diagram of dCas9-SAM protein structure simulation (Cyans: dCas9. Red: VP64. Yellow: MS2. Brown: p65. Purple: HSF1). B: Western blot identification of dCas9-SAM protein expression in positive cells (1: Control of HIEC6 cells; 2-14: HIEC6-dCas9-SAM positive cells)

图2 HIEC6-dCas9-SAM单克隆细胞株的Western blot鉴定

Fig. 2 Western blot identification of HIEC6-dCas9-SAM monoclonal cells

图3 HIEC6-dCas9-SAM细胞的免疫荧光检测（200×）

Fig. 3 Immunofluorescence detection of HIEC6-dCas9-SAM monoclonal cells (200×)

图4 荧光报告系统检测HIEC6-dCas9-SAM细胞的CRISPR激活效率A：CRISPRa荧光报告质粒及对照质粒示意图（TRE：TetO响应元件；TetO gRNA：可靶向TRE募集dCas9-SAM并驱动GFP表达； Empty gRNA：空白对照）；B：荧光显微镜观察GFP蛋白表达情况（Empty：对照质粒；CRISPRa Rporter：CRISPRa荧光报告质粒）；C：流式检测激活效率（sgRNA expression/BFP：报告质粒和对照质粒转导进细胞，表达蓝色荧光蛋白；GFP：TetO gRNA靶向TRE募集dCas9-SAM进行转录激活，表达绿色荧光蛋白）

Fig. 4 CRISPR activation efficiency of HIEC6-dCas9-SAM cells detected by fluorescent reporter systemA: Schematic diagram of CRISPRa fluorescent reporter plasmid and control plasmid (TRE: TetO response element.TetO gRNA: Targeting TRE to recruit dCas9-SAM and drive GFP expression. Empty gRNA: Blank control gRNA). B: Observation of GFP protein expression by fluorescence microscope (Empty: control plasmid. CRISPRa Reporter: CRISPR activation reporter plasmid). C: Detection of activation efficiency by flow cytometry (sgRNA expression/BFP: report plasmid and control plasmid transduced into cells, expressing blue fluorescent protein. GFP: TetO gRNA targeted TRE recruits dCas9-SAM for transcriptional activation and expression of green fluorescent protein)

图5 qPCR、Western blot检测HIEC6-dCas9-SAM细胞对靶基因的转录激活效果A：特异性sgRNA慢病毒载体示意图；B：sgAPN、sgSLC35A1慢病毒载体测序结果；C：qPCR检测APN基因相对表达量；D：qPCR检测SLC35A1基因相对表达量（****P<0.000 1表示单因素方差分析两组间基因相对表达量差异极显著）；E：Western blot检测CRISPR激活后的APN蛋白表达量；F：Western blot检测CRISPR激活后的SLC35A1蛋白表达量（1：空白对照组；2：HIEC6细胞；3：1# HIEC6-dCas9-SAM细胞；4：4# HIEC6-dCas9-SAM细胞）

Fig. 5 qRCR and Western blot detection of transcriptional activation of target genes in HIEC6-dCas9-SAM cellsA: Schematics of specific sgRNA lentiviral vector. B: Sequencing result of sgAPN and sgSLC35A1 lentiviral vector. C: Relative expression of APN gene detected by qPCR. D: Relative expression of SLC35A1 gene detected by qPCR (****P<0.000 1 indicates that the relative expression of gene was significantly different between the two groups in one-way ANOVA). E: APN protein expression after CRISPR activation by Western blot. F: SLC35A1 protein expression after CRISPR activation by Western blot (1: Blank;2: HIEC6 cell; 3: 1# HIEC6-dCas9-SAM cell; 4: 4# HIEC6-dCas9-SAM cell)

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具备高效CRISPR协同激活活性的HIEC6-dCas9-SAM稳转细胞株构建

Construction of HIEC6-dCas9-SAM Transgenic Cell Line with Highly-efficient CRISPR Synergistic Activation Properties

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