Zeocin

To perform CRISPR/Cas9-genome editing, 200,000 HEK293-T cells were transfected with 500 ng of all-in-one Cas9-nuclease and gRNA plasmid (pX330S-2+gRNA-S663), 100 pmol of ssODN homology-directed repair (HDR) donor complementary to the anti-sense strand (5′.CGCCGCATCGGCATCTTCGGGCAGGATGAGGACGTGACGTCAAAAGCTTTCACAGGCCGGGAGTTTGATGAACTCAACCCCgCgGCCCAGCGAGACGCCTGCCTGAACGCCCGCTGTTTTGCTCGAGTTGAACCCTCCC.3′; mutated nucleotides are in lowercase), and 50 ng of pcDNA4 (Invitrogen, V1020-20) Zeocin selectable plasmid. From 24 h after transfection, 1 µM SCR7 pyrazine (Sigma, SML1546) and 200 µg/mL Zeocin (Gibco, R25001) were added to promote HDR at Cas9-induced DSB^{56 (link)}, and to select transfected cells respectively. Single-cell-derived colonies were screened for expected base substitution thanks to polymerase chain reaction (PCR) amplification of a 281 base pairs (bp) genomic sequence spanning targeted locus of ATP2A2 (forward primer 5′.ACCAATCTGACCTTCGTTGG.3′ and reverse primer 5′.GAGGGTTCAACTCGAGCAAA.3′) followed by SacII digestion (NEB, R0157) and Sanger sequencing. ssODN-mediated HDR was design to insert a SacII restriction site that give two fragments of 227 and 54 bp after digestion that does not exist in wild-type genome. A validated clone was kept to generate HEK293-T SERCA2[S663A] cell line used for this study.

To establish Rep-EGFP or Rep-mHRP stable clone cell lines, BHK-21 cells were transfected with pRep-EGFP or pRep-mHRP. After 24 hours of transfection, the cells were seeded into 96-well plates at limiting dilution and incubated in culture medium containing 400 μg/ml Zeocin^™ (Invitrogen). Then, the plates were incubated at 37°C in a humidified CO₂ incubator until a single colony could be identified in each well. Then, the colonies were subcultured from the wells into larger vessels. The Rep-EGFP or Rep-mHRP monoclonal cell lines were verified by selection of the EGFP⁺ or HRP⁺ cells after pCre transfection. To establish F-DENPADS and H-DENPADS cell lines, pSen-Cre (Fig 1) was transfected into Rep-EGFP or Rep-mHRP stable cell lines, and cells were cultured at limiting dilution as described above. Then, the cells were incubated in culture medium containing 400 μg/ml Zeocin^™ and 800 μg/ml Geneticin (Gibco). F-DENPADS cell lines and H-DENPADS cell lines were verified by selection of the EGFP⁺ or HRP⁺ cells after DENV-2 infection, and the expression of Sen-Cre was verified by western blot.

To test the target selectivity of α5-SOP002, a stable cell line of HEK293 cells expressing α5β2γ2 subunits of the GABA_AR was developed using the previously established method based on antibiotic selection (Brown et al., 2016 (link)). HEK293 cells (2 × 106) were transfected using Lipofectamine LTX (catalog no. 15338–100, Invitrogen) with the α5 pcDNA3.1(+) construct, incorporating the G418 disulfate (Neomycin) resistance gene and β2 pcDNA3.1(+) construct, incorporating the Zeocin resistance gene. Cells were subsequently plated at the ratios of 1:3, 1:5, 1:7, 1:10, 1:15, and 1:20, and selected with G418 (Neomycin; catalog no. G5013, Sigma–Aldrich) and Zeocin (catalog no. R25001 Gibco) antibiotics (both at 800 μg/ml) until colonies were formed. After 7 days, ~5–20 single colonies were selected and gradually scaled up. The clone expressing the highest level of GABA_AR α5 and β2 subunits, as well as the previously established α2β2-HEK293 (Brown et al., 2016 (link)) stable cell line were further transfected with the γ2 pcDNA3.1(+) construct, incorporating the Hygromycin resistance gene, to produce triple cell lines. The expression of all three subunits was characterized by immunoblotting and immunocytochemistry. The α1β2γ2-HEK293 was characterized previously (Fuchs et al., 2013 (link)).

The xynA gene from P. citrinum FERM P-15944 was synthesized with codon optimization for expression in P. pastoris based on the nucleotide database (GenBank: accession no. AB198065.1) [21 (link)] and introduced into the pUC57-Kan vector by GenScript (Piscataway, NJ, USA). The P. pastoris expression kit, including the P. pastoris strain X-33, constitutive expression vector (pGAPZα A), methanol-inducible expression vector (pPICZα A) and Zeocin were all from Invitrogen (Carlsbad, CA, USA). All restriction enzymes and Phusion High-Fidelity DNA polymerase were from New England BioLabs (Beverly, MA, USA). Birchwood xylan was from Sigma-Aldrich (St Louis, MO, USA), Azo-xylan was from Megazyme (Wicklow, Ireland).
Pichia pastoris was cultured in YPD medium 1% (w/v) yeast extract, 2% (w/v) bacteriological peptone and 2% (w/v) dextrose at 30 °C with shaking at 200 rpm. The transformants were selected on YPDS plates (YPD with 1 M sorbitol) containing 100–2000 µg/mL Zeocin. The pGAPZα A transformants were chosen for preliminary selection on YP (YPD without dextrose) with 0.2% (w/v) Azo-xylan.
Escherichia coli DH5α (Gibco) was used for vector propagation and was grown in a Luria-Bertani (LB) medium at 37 °C with either kanamycin (50 µg/mL) to select for pUC57-Kan or Zeocin (25 µg/mL) to select for pGAPZα A and pPICZα A transformants.