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Construction of an Inducible CRISPR/Cas9 System for CXCR4 Gene and Demonstration of its Effects on MKN-45 Cells

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Abstract

The CRISPR/Cas9 system is an effective tool for gene editing. However, this conventional expression system cannot control the timing of gene editing and does not utilize resistance screening markers. Therefore, carrying out CRISPR/Cas9 experiments is extremely inconvenient. Our aim is to develop an inducible lentiviral vector-based gene-editing system for C-X-C chemokine receptor 4 (CXCR4) by CRISPR/Cas9, and to demonstrate its function in MKN-45 cell. The DNA fragments of Blasticidin and T2A-GFP were produced using the lenti-Cas9-BLAST and PX458 plasmids as templates. The PCR products were harvested and cloned into the lenti-guide-puro plasmid to yield the lenti-guide-BLAST-GFP plasmid. Three double-stranded guide RNA (gRNA) sequences targeting the exon 2 of CXCR4 gene were designed online (http://crispr.mit.edu), synthesized, and recombined into the lenti-guide-BLAST-GFP plasmid, to yield the lenti-guide-BLAST-GFP-gRNA plasmid. The pCW-Cas9 and lenti-guide-BLAST-GFP-gRNA plasmids were packaged with lentiviral vectors, which were then transfected into MKN-45 cells, to identify the CXCR4 gene-editing effects using the T7 endonuclease 1 (T7E1) and Western blot assays. The lenti-guide-BLAST-GFP and lenti-guide-BLAST-GFP-gRNA plasmids were successfully constructed and packaged, to yield lentiviral particles. Transfection of the pCW-Cas9 and lenti-guide-BLAST-GFP-gRNA viral vectors could decrease the expression of CXCR4 protein, and lead to gene editing in MKN-45 cells. The efficiencies of gRNA-1, gRNA-2, and gRNA-3 were 45.6%, 53.6%, and 56.7%, respectively. Furthermore, the chemotactic efficiency of the dual viral vector-infected MKN-45 cells was significantly decreased in response to SDF-1. The numbers of migratory cells in the lower chamber of the transwell system were 30.0 ± 0.23, 29.7 ± 1.55, 28.2 ± 1.11 and 36.1 ± 2.00 cells per field (400×) for gRNA-1, gRNA-2, gRNA-3 and the control, respectively (P < 0.05). We constructed an inducible CXCR4 gene-editing, dual-vector CRISPR/Cas9 system, which could induce CXCR4 gene editing in MKN-45 cells in a doxycycline-dependent manner and thus reduce the migration of MKN-45 cells.

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Fig. 1
Fig. 2: Dual viral vector infection in MKN-45 cells.
Fig. 3: CRISPR/Cas9-mediated CXCR4 gene editing in MKN-45 cells.
Fig. 4: CRISPR/Cas9-mediated decrease in CXCR4 protein expression in MKN-45 cells.
Fig. 5: CRISPR/Cas9-mediated decrease in MKN-45 cell migration in response to SDF-1.

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Abbreviations

CXCR4:

C-X-C chemokine receptor type 4

SDF-1:

stromal cell-derived factor-1

MKN-45:

gastric adenocarcinoma MKN-45 cells

WHIM:

warts, hypogammaglobulinemia, infections, and myelokathexis syndrome

ZFN:

zinc finger nuclease

TALEN:

transcription activator-like effector nuclease

CRISPR:

clustered regularly interspaced short palindromic repeats

Cas9:

CRISPR-associated nuclease 9

gRNA:

guide RNA

DMEM:

Dulbecco’s modified Eagle’s medium

BLAST:

blasticidin

GFP:

green fluorescent protein

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Acknowledgements

The authors would like to thank the staff of the Chongqing Key Laboratory of Pediatrics for their valuable contributions to this project.

Funding

This work was financially supported by the National Natural Science Foundation of China [No. 30700785] and the Chongqing Health and Family Planning Commission [No. 2015MSXM040]. The funders had no role in the design of the study, data collection and analysis, decision for publication, or preparation of the manuscript.

Author Contributions

Y.P. and S.W. carried out the molecular laboratory work, participated in the data analysis, carried out the sequence alignments, participated in the design of the study and drafted the manuscript; T.Y. carried out the statistical analyses; and X.T. and F.C. conceived, designed, and coordinated the study and helped draft the manuscript. All authors gave final approval for publication.

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Authors and Affiliations

  1. Department of Anesthesiology, Children’s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, No. 136, 2nd Zhongshan Rd, Yuzhong District, Chongqing, China

    Yanhua Peng, Taobo Yang, Xixi Tang, Fei Chen & Shouyong Wang

  2. Department of Anesthesiology, People’s Hospital of Deyang City, Taishan North Road 173, Deyang, 618000, China

    Yanhua Peng

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  1. Yanhua Peng
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Correspondence to Shouyong Wang.

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Peng, Y., Yang, T., Tang, X. et al. Construction of an Inducible CRISPR/Cas9 System for CXCR4 Gene and Demonstration of its Effects on MKN-45 Cells. Cell Biochem Biophys 78, 23–30 (2020). https://doi.org/10.1007/s12013-019-00898-x

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