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 W...

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

10.1007/s12013-019-00898-x