Puromycin resistance

To generate A549 cells overexpressing, UBCv1 was amplified by PCR using primers 5′ GACGGATCCATGGATTACAA -3′ (Fw) and 5′- GCGCTCTAGA TTACTCATCATC -3′ (Rv). The PCR product was subcloned into the commercially lentiviral expression vector pLVX-Puro (Clontech, Mountain View, CA, USA). Lentiviral particles were produced by transfection of HEK293T cells (seeded in 60 mm dishes) with 5 μg of pLVX, 3 μg of psPAX2 packaging plasmid (Addgene 12260, Watertown, MA, USA), and 3 μg of vesicular stomatitis virus-G glycoprotein using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). Supernatants containing the lentivirus were harvested at 48 and 72 h post transfection, 0.45 μM-filtered, and used to transduce A549 cells, which were subsequently selected for puromycin resistance (Life Technologies, 5 μg/mL).

Ready-to-use lentiviral particles were purchased from Sigma-Aldrich. hCMEC/D3 cells were transduced first by lentiviral particles expressing Cas9 and blue fluorescent protein (BFP) as a transduction marker (LVCAS9BST, Sigma-Aldrich). Cas9-expressing cells were then selected with blasticidin antibiotic at 50 μg/mL for 6 days (Life technologies). Cas9-expressing hCMEC/D3 cells were each transduced separately by lentiviral particles expressing sgRNAs and selected by puromycin resistance at 10 μg/mL for 6 days (Life technologies). sgRNA sequences are listed in Table 2.Table 2

List of sgRNAs

sgRNA name	Gene ID	Sanger Clone ID	PAM sequence	DNA target sequence
TFRC sgRNA	TFRC	HS5000003796	CGG	TGATCATAGTTGATAAGAACGG
CAV1 sgRNA	CAV1	HS0000173752	CGG	GACGTAGATGGAATAGACA
CLTC sgRNA	CLTC	HS5000032727	TGG	TAACTACAGGAAGTCGACTTGG

To generate monoclonal KO cell populations, Cas9-gRNA-expressing hCMEC/D3 were sorted (FACSAria II, BD Biosciences) using BFP into single clones and subsequently expanded.