CRISPR-Mediated Knockout of Gnptab in CHO Cells

Knockout (KO) of GlcNAc-1-phosphate transferase subunits alpha and beta (Gnptab) gene was carried out in selected CHO clones that stably express the lysosomal enzymes of interest. Cells were seeded at a density of 0.5 × 10⁶ cells/mL in T25 flasks 24 h prior to transfection, and ∼2 × 10⁶ cells and 1 μg each of plasmid DNA of Cas9-GFP and gRNA were used for electroporation. 48 h after electroporation, cells with GFP expression were enriched by FACS. After culturing for 1 week, cells were single cell sorted by FACS into 96-wells. KO clones with desired mutations were identified using a rapid and efficient screening method, Indel Detection by Amplicon Analysis (IDAA), as previously described (Yang et al., 2015a (link)). Final clones were verified by Sanger sequencing. On average 2–5 clones with frameshift mutations were selected from each targeting event. The full list of CRISPR gRNA design and PCR primers used is listed elsewhere (Tian et al., 2019 (link)).