Synthesis a New Viral Base Vector Carrying Single Guide RNA (sgRNA) and Green Florescent Protein (GFP)

Abstract

CRISPR/Cas9 system is a powerful gene editing tool in vivo and in vitro. Currently, CRISPR/Cas9 delivery cells or tissue with different vehicles are available, and Adeno- associated virus (AAV) in one of them. Due to AAV packaging size limitation, AAV base vectors that carry CRISPR/Cas9 system do not have florescent tag like GFP for simple detection and navigation of cells, containing AAV. The aim of this study was to modify and synthesis AAV base vector for CRISPR/cas9 system containing sgRNA and GFP.Px602 plasmid was double digested with NcoI and HindIII restriction enzyme. Gfp gene was amplified from px458 plasmid. Linear digested px602 and amplified Gfp gene were ligated together. After transformation and colony PCR on white colonies, plasmid was extracted and transfected to HEK-293 cell line. Gfp expression was monitored by florescent microscopy. After transfection of modified plasmid, florescent microscopy of HEK-293 cells showed shining green florescent cells, which indicate that Gfp gene, was replaced in the correct place according to our design.We modified an AAV base vector carrying CRISPR/Cas9 system, and synthesized a new vector carrying Gfp gene and sgRNA that can be packaged as reporter AAV for navigation and detection of cells, containing AAV.