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

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

Abstract

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

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.

Figures/Tables 6

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

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)

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

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

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)

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)

References 36

1	Cong L, Ann Ran F, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems [J]. Science, 2013, 339(6121): 819-823.
2	Jiang FG, Doudna JA. CRISPR-Cas9 structures and mechanisms [J]. Annu Rev Biophys, 2017, 46: 505-529.
3	Shields RC, Walker AR, Maricic N, et al. Repurposing the Streptococcus mutans CRISPR-Cas9 system to understand essential gene function [J]. PLoS Pathog, 2020, 16(3): e1008344.
4	Zhu YM. Advances in CRISPR/Cas9 [J]. BioMed Res Int, 2022, 2022(1): 9978571.
5	Gilbert LA, Horlbeck MA, Adamson B, et al. Genome-scale CRISPR-mediated control of gene repression and activation [J]. Cell, 2014, 159(3): 647-661.
6	Chavez A, Tuttle M, Pruitt BW, et al. Comparison of Cas9 activators in multiple species [J]. Nat Methods, 2016, 13(7): 563-567.
7	Konermann S, Brigham MD, Trevino AE, et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex [J]. Nature, 2015, 517(7536): 583-588.
8	Heidersbach AJ, Dorighi KM, Gomez JA, et al. A versatile, high-efficiency platform for CRISPR-based gene activation [J]. Nat Commun, 2023, 14(1): 902.
9	Han K, Jeng EE, Hess GT, et al. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions [J]. Nat Biotechnol, 2017, 35(5): 463-474.
10	Pan JM, Zhang M, Dong LQ, et al. Genome-Scale CRISPR screen identifies LAPTM5 driving lenvatinib resistance in hepatocellular carcinoma [J]. Autophagy, 2023, 19(4): 1184-1198.
11	Feng F, Zhu YK, Ma YL, et al. A CRISPR activation screen identifies genes that enhance SARS-CoV-2 infection [J]. Protein Cell, 2022, 14(1): 64-68.
12	Awwad SW, Serrano-Benitez A, Thomas JC, et al. Revolutionizing DNA repair research and cancer therapy with CRISPR-Cas screens [J]. Nat Rev Mol Cell Biol, 2023, 24(7): 477-494.
13	Yang LP, Sheets TP, Feng Y, et al. Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform [J]. Sci Adv, 2024, 10(7): eadj2445.
14	Danziger O, Patel RS, DeGrace EJ, et al. Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor [J]. PLoS Pathog, 2022, 18(4): e1010464.
15	Rahman MM, Tollefsbol TO. Targeting cancer epigenetics with CRISPR-dCAS9: principles and prospects [J]. Methods, 2021, 187: 77-91.
16	Beaulieu JF, Ménard D. Isolation, characterization, and culture of normal human intestinal crypt and villus cells [J]. Methods Mol Biol, 2012, 806: 157-173.
17	Pászti-Gere E, Pomothy J, Jerzsele Á, et al. Exposure of human intestinal epithelial cells and primary human hepatocytes to trypsin-like serine protease inhibitors with potential antiviral effect [J]. J Enzyme Inhib Med Chem, 2021, 36(1): 659-668.
18	Perreault N, Beaulieu JF. Use of the dissociating enzyme thermolysin to generate viable human normal intestinal epithelial cell cultures [J]. Exp Cell Res, 1996, 224(2): 354-364.
19	Pageot LP, Perreault N, Basora N, et al. Human cell models to study small intestinal functions: recapitulation of the crypt-villus axis [J]. Microsc Res Tech, 2000, 49(4): 394-406.
20	Tremblay E, Auclair J, Delvin E, et al. Gene expression profiles of normal proliferating and differentiating human intestinal epithelial cells: a comparison with the Caco-2 cell model [J]. J Cell Biochem, 2006, 99(4): 1175-1186.
21	Guezguez A, Paré F, Benoit YD, et al. Modulation of stemness in a human normal intestinal epithelial crypt cell line by activation of the WNT signaling pathway [J]. Exp Cell Res, 2014, 322(2): 355-364.
22	Juntarachot N, Sunpaweravong S, Kaewdech A, et al. Characterization of adhesion, anti-adhesion, co-aggregation, and hydrophobicity of Helicobacter pylori and probiotic strains [J]. J Taibah Univ Med Sci, 2023, 18(5): 1048-1054.
23	Jiang YH, Zhang GQ, Li LT, et al. Transcriptomic analysis of PDCoV-infected HIEC-6 cells and enrichment pathways PI3K-Akt and P38 MAPK [J]. Viruses, 2024, 16(4): 579.
24	Jiang YH, Zhang GQ, Li LT, et al. A novel host restriction factor MRPS6 mediates the inhibition of PDCoV infection in HIEC-6 cells [J]. Front Immunol, 2024, 15: 1381026.
25	Joung J, Kirchgatterer PC, Singh A, et al. CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity [J]. Nat Commun, 2022, 13(1): 1606.
26	巩琦凡. 基于CRISPRa胃癌耐药相关基因筛选及功能研究 [D]. 保定: 河北大学, 2023.
	Gong QF. Screening and functional study of drug resistance-related genes in gastric cancer based on CRISPRa [D]. Baoding: Hebei University, 2023.
27	Zhu SY, Liu Y, Zhou Z, et al. Genome-wide CRISPR activation screen identifies candidate receptors for SARS-CoV-2 entry [J]. Sci China Life Sci, 2022, 65(4): 701-717.
28	Clark T, Waller MA, Loo L, et al. CRISPR activation screens: navigating technologies and applications [J]. Trends Biotechnol, 2024, 42(8): 1017-1034.
29	Sastry L, Xu Y, Duffy L, et al. Product-enhanced reverse transcriptase assay for replication-competent retrovirus and lentivirus detection [J]. Hum Gene Ther, 2005, 16(10): 1227-1236.
30	Sandoval-Villegas N, Nurieva W, Amberger M, et al. Contemporary transposon tools: a review and guide through mechanisms and applications of Sleeping beauty, piggyBac and Tol2 for genome engineering [J]. Int J Mol Sci, 2021, 22(10): 5084.
31	Rostovskaya M, Fu J, Obst M, et al. Transposon-mediated BAC transgenesis in human ES cells [J]. Nucleic Acids Res, 2012, 40(19): e150.
32	Eckermann KN, Ahmed HMM, KaramiNejadRanjbar M, et al. Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species [J]. Insect Biochem Mol Biol, 2018, 98: 16-24.
33	Vargas JE, Chicaybam L, Stein RT, et al. Retroviral vectors and transposons for stable gene therapy: advances, current challenges and perspectives [J]. J Transl Med, 2016, 14(1): 288.
34	Chong ZS, Ohnishi S, Yusa K, et al. Pooled extracellular receptor-ligand interaction screening using CRISPR activation [J]. Genome Biol, 2018, 19(1): 205.
35	Zhou J, Wan J, Gao XW, et al. Dynamic m(6)a mRNA methylation directs translational control of heat shock response [J]. Nature, 2015, 526(7574): 591-594.
36	Moreno-Mateos MA, Vejnar CE, Beaudoin JD, et al. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo [J]. Nat Methods, 2015, 12(10): 982-988.

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