Fig. 1

A CRISPR HDR-based strategy was designed to integrate MetRS^L274G into the CLYBL safe harbor locus of human H4 neuroglioma cells, select for single clones, and excise the selection cassette to leave minimally modified cells stably expressing MetRS^L274G (a). On-target integration and successful excision of selection cassette was confirmed using PCR primer-pairs designed to encompass a flanking and internal region of the donor plasmid (b). Expression of functional MetRS^L274G was validated by FUNCAT—H4.WT or MetRS^L274G-expressing cells were cultured with or without 4 mM ANL. Only those expressing MetRS^L274G and exposed to ANL-containing media displayed abundant labeling with Cy5-DBCO (c). Scale bars are 10 µm. Uncropped gel from (b) is shown in Additional file Fig. S1