CRISPR/Cas9 gene editing for the creation of an MGAT1-deficient CHO cell line to control HIV-1 vaccine glycosylation
Fig 2
Flow chart of MGAT1 gene editing and cell line selection strategy.
(A) A plasmid containing the Cas9 nuclease, tracrRNA, and a gRNA sequence was electroporated into suspension adapted CHO-S cells. (B) Twenty-four hours following transfection, the cells were distributed into 96-well tissue culture plates at a density of 0.5 cells/well. (C) Between 12 and 15 days later, wells with 20% or greater confluency were transferred to 24-well plates. (D) After 5 days of growth in 24-well plates, a 0.2-mL aliquot was removed from each well, and cells were tested for the ability to bind fluorescein-labeled GNA. (E) GNA-binding cells were then expanded to shake flasks, and cell lines were transiently transfected with a gene encoding A244-rgp120. The cell culture supernatants were then collected after 5 days and tested for binding of gp120 to the prototypic glycan-dependent, broadly neutralizing monoclonal antibody PG9. This representative plot (F) is shown for demonstrative process purposes only. A detailed plot of this data is show in Fig 4A. (G) The gene encoding MGAT1 was sequenced from GNA-binding cell lines that exhibited robust growth and the ability to secrete PG9-binding gp120. The specific mutations induced by NHEJR were determined by Sanger sequencing. Cas9, CRISPR-associated protein 9; CHO, Chinese hamster ovary; FIA, fluorescence immunoassay; GNA, Galanthus nivalis lectin; gRNA, guide RNA; MGAT1, Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase; NHEJR, nonhomologous end joining repair; rgp120, recombinant gp120; tracrRNA, trans-activating CRISPR RNA.