PRODH/POX Gene Knockout Using CRISPR

The sgRNAs for PRODH/POX (CRISPR All-In-One Non-Viral Vector with spCas9) were ordered by ABM Company (Richmond, VA, Canada). The vector with expression construct (directed against PRODH1 isoform) was transformed into Escherichia coli DH5α and grown in Luria–Bertani (LB) media supplemented with 100 µg·mL⁻¹ ampicillin at room temperature for 24 h, as described previously [60 (link)]. The targeted plasmid was extracted by a plasmid DNA purification kit (Nucleobond Xtra Midi/Maxi, MACHERY-NAREL GmbH, Düren, Germany). After being precipitated by isopropanol, the purified samples were washed by 70% ethanol solution and then followed by a DNA cleaning-up step by a GeneMATRIX Basic DNA Purification Kit (EURX, E3545-01 protocol 1, Gdansk, Poland). The purified DNA concentration was estimated by NanoDrop™ 2000/2000c Spectrophotometers (Thermo Fisher Scientific, Waltham, MA, USA).