CRISPR-Mediated Knock-Out of SLC35A1

For knocking out the expression of the sialic acid transporter SLC35A1 in HEK293T wild-type, HEK293T-AGA-knockout [19 (link)], and HeLa wild-type cells, the guide RNAs (gRNAs) were designed with the E-Crisp design tool (DKFZ, Heidelberg, Germany) and cloned via BbsI into pSpCas9(BB)-2A-Puro (PX459, Addgene plasmid #48139, a kind gift of Feng Zhang) [20 (link)]. The gRNA sequences are shown in Table 1. Twenty-four hours before transfection, cells were seeded onto 6-well plates. For transfections, 2.5 µg genome editing plasmid was transfected using MACSfectin™ (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s protocol. After 24 h, the cells received a culture medium containing puromycin (2 µg/mL) for 24 h to eliminate untransfected cells. Thereafter, the cells were counted and seeded into 96-well plates (1 cell/well). Wells containing single-cell clones were expanded and used for further analysis. For analysis, cells were lysed with the Phire™ animal tissue direct PCR kit (Thermo Fisher Scientific, Dreieich, Germany). Genomic DNA was PCR amplified. The knockout was confirmed by sequencing the PCR products (Microsynth Seqlab, Göttingen, Germany). Primer sequences are shown in Table 1. Lack of protein expression was confirmed by immunofluorescence staining for SLC35A1.