Construction Of An Gene Editing Lentiviral Vector System For C-X-C Chemokine Receptor 4 Using CRISPR/Cas9

Objective: To develop an inducible lentiviral vector-based gene editing system for C-X-C chemokine receptor 4(CXCR4)using the CRISPR/Cas9 technique and to demonstrate its gene editing function in MKN-45 cells.Methods: Blasticidin and T2A-GFP DNA fragments were produced using lenti-Cas9-BLAST and PX458 plasmids as templates.The PCR products were harvested by agarose gel electrophoresis and cloned into the lenti-guide-puro plasmid to yield the lenti-guide-BLAST-GFP plasmid.Three double-stranded guide RNA(gRNA)sequences targeting exon 2 of the CXCR4 gene were designed online(http://crispr.mit.edu)and synthesized,followed by recombination 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 and transfected into MKN-45 cells in sequential order to demonstrate the CXCR4 gene-editing effects using the T7 endonuclease 1(T7E1)assay.Results: Gene sequence reports suggested the successful construction and packaging of the lenti-guide-BLAST-GFP and lenti-guide-BLAST-GFP-gRNA plasmids to yield lentiviral particles.Transfection of the pCW-Cas9 and lenti-guide-BLAST-GFP-gRNA viral vectors decreased CXCR4 protein expression and led to gene editing in MKN-45 cells,and the efficiency of gRNA-1,gRNA-2,and gRNA-3 was 45.6%,53.6%,and 56.7% respectively.Western blotting experiment confirmed that the expression of CXCR4 protein was significantly reduced.Furthermore,the chemotactic efficiency of the dual viral vector-infected MKN-45 cells was significantly decreased in response to SDF-1,and the numbers of migratory cells in the lower chamber of the transwell system were 30.03± 0.23,29.73± 1.55,28.20± 1.11 and 36.13± 2.00 cells per field(400×)for g RNA-1,gRNA-2,gRNA-3 and the control,respectively(P<0.05).Conclusion: We constructed an inducible CXCR4 gene-editing,dualvector CRISPR/Cas9 system that can induce CXCR4 gene editing in MKN-45 cells in a doxycycline-dependent manner.