FIG 3.

Epitope-tagging of GP133 by homology-directed repair. (A) Diagram of the homology repair template (HRT) used to introduce the HA-2A-GPT tag to the 3′ end of GP133. Modifications to the GP133 coding sequence (CDS) were as follows: amino acids overlapping the CRISPR site were codon optimized (black arrow) and sequences encoding a 3' HA-tag, a glycine-serine-glycine (GSG) spacer, a 2AT peptide, and a flippase recognition target (FRT)-flanked GPT were added. (B) PCR amplification of passaged virus-containing media from transfection/infection experimentsusing gRNA plasmids targeting the 3′ end of GP133, with or without the HRT. The initial transfection/infections were carried out in the presence or absence of the DNA ligase IV inhibitor SCR7, and the virus was passaged with or without GPT selection. (C) PCR amplification of GP133 from viruses isolated either immediately after transfection/infection or after a single passage in media containing mycophenolic acid xanthine to enrich for recombinant viruses. (D) Immunoblots of cell lysates from mock-infected GPL cells and cells infected with GPCMV GP133-HA-2A-GPT or its parental virus, RFP GPCMV. Primary antibodies against the HA tag, 2A peptide, GPCMV gB, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used.