An improved strategy for CRISPR/Cas9 gene knockout and subsequent wildtype and mutant gene rescue
Fig 1
Construction of pLentiCrispr-V2-mOrange (V2mO), its lentiviral production in HEK293T and transduced mOrange expression in GC cell lines AGS and GT5.
A. V2mO plasmid construct, with mOrange cistronically inserted between Cas9 and puromycin cDNAs. Between the original pLentiCrispr-Cas9-V2 plasmid (top) and the V2mO plasmid (bottom) are shown the overlapping sequences and restriction enzymes for the construction. B. HEK293T cells expressed mOrange in lentiviral production, which enables estimation of transformation efficiency and viral titer. C & D. Target cell lines AGS (C) and GT5 (D) were transduced with lentiviral V2mO-RhoA.g5 after puromycin selection and mOrange sorting. E. Cell line GT5 was transduced with lentiviral V2mO-Gli1.g4 after puromycin selection and mOrange sorting. F & G. Cell lines AGS (F) and GT5 (G) were transduced with lentiviral V2mO-Gal3.g1 after puromycin selection and mOrange sorting.