CRISPR/Cas9-mediated knockout of GRP78
For lentiviral experiments, three sgRNA sequences targeting GRP78 were
selected based on enrichment scores from the whole genome CRISPR screens
and cloned into the lentiCRISPR v2 (Addgene plasmid no. 52961) according
to the protocol provided by the Zhang Lab (Zuo et al., 2017). A complete
list of CRISPR sequences can be found in Online Supplementary Table 4.
Lentivirus was generated by co-transfecting HEK293T cells with
lentiCRISPR v2, envelop plasmid pMD2.G (Addgene plasmid no. 12259), and
packaging plasmid psPAX2 using Lipofectamine 2000 (Invitrogen, Carlsbad,
CA, USA), Viral supernatants were collected 48 hours following
transfection and used to infect HEK293T cells.
Puromycin
selection (3μM, HY-15695, MCE) was performed for 3 to 4 days establish
the stable cell lines.