Stellar cells

pSCRPSY-Tag-RFP-ACE2 (kindly provided by John Schoggins) was used for lentivirus production as described above, and Huh7 cells were transduced and selected for using 0.5 ug/ml Blasticidin. ACE2 expression was confirmed via RFP expression. sgRNAs with highest scores in CRISPR KO screen were ordered as forward and reverse oligos for creation of stable KO cell lines.
Oligonucleotides were denatured for 5 minutes at 99°C in TE buffer and then slowly adapted to room temperature (RT) and assembled with pLentiCRISPRv2 vector using Golden Gate cloning. Plasmids were transformed in Stellar cells (Takara, Kusatsu, Shiga, Japan) and prepped for Sanger sequencing and lentivirus production. ACE2-expressing Huh7 cells were transduced with pLentiCRISPRv2 containing sgRNAs for top scoring hits and selected with 0.25 ug/ml puromycin. Bulk KO of FKBP8, TMEM41B, and MINAR1 was verified using Sanger sequencing and western blot.

A DNA segment encompassing the MCTS1 exon 2 and 3 region (ChrX: 120604950 to ChrX: 120606870 region, GRCh38 reference, release 13) was amplified from genomic DNA extracted from whole-blood samples from a healthy control. It was inserted between the XhoI and BamHI sites of the pSPL3 vector. The sequence was then mutated to encode the variant of P3. Plasmids containing wild-type (WT) and mutant MCTS1 exon 2-3 regions were then used to transfect COS-7 cells. After 24 hours, total RNA was extracted and reverse-transcribed. The cDNA products were amplified with primers binding to the flanking HIV-TAT sequences of the pSPL3 vector and ligated into the pCR^™4-TOPO^® vector (Invitrogen). Stellar^™ cells (Takara) were transformed with the resulting plasmids. Colony PCR and sequencing with primers binding to the flanking HIV-TAT sequences of pSPL3 were performed to assess the splicing products transcribed from the WT and mutant alleles.