mRNA analysis using patient-derived lymphoblastoid cell lines

To generate patient-derived LCLs, lymphocytes were transformed with the Epstein-Barr virus at a core facility run by Tokyo Medical Dental University. The origin of the LCL cells was identified as patients by partial sequencing of the genome. LCLs were cultured in RPMI1640 medium (ThermoFisher Scientific, Waltham, MA) supplemented with 15% fetal bovine serum (FBS; ThermoFisher Scientific), 2mM l-glutamine (ThermoFisher Scientific), and 1% penicillin/streptomycin (ThermoFisher Scientific) at 37 °C in an atmosphere of 5% CO₂. Cells were tested routinely for mycoplasma contamination. A plasmid for CAG promoter insertion genome editing⁵⁰ was constructed as shown in Supplementary Fig. 4A, B. The donor template, which comprised the flanking micro-homology arms, gRNA target site, and the donor sequence, were sub-clone and inserted into the single CRISPR/Cas9 vector (pX601, addgene #61591). gRNAs were designed (Supplementary Fig. 4C) and T7E1 assay (Supplementary Fig. 4D) were performed as the manufacturer’s instructions (New England Biolabs, Ipswich, MA). In brief, PCR products amplified using genomic DNA were denatured at 95 °C for 5 min, reannealed, and incubated with T7 Endonuclease I (New England Biolabs) at 37 °C for 30 min. The reaction products were resolved by electrophoresis in 2% agarose gel. DNA fragments were analyzed using ImageJ. The indel efficiency was calculated as 100 × (1 − (1−cleaved band intensity/total band intensities)1/2). The donor sequence included a CMV promoter (from pCAG-Neo, Wako, Osaka, Japan) for in-frame insertion upstream of the EYS start codon. A plasmid for mutation replacement genome editing was constructed as shown in Supplementary Fig. 4E, F. The donor template, which comprised the flanking micro-homology arms, gRNA-1 target site or gRNA-4 target site, and the donor sequence, were sub-cloned and inserted into the vector (pX601) using a DNA ligation kit (Clontech, Mountain View, CA). To avoid repeated cleavage after mutation replacement, mutations were introduced in the flanking gRNA target sites within the donor template. The mutations introduced in the 5′ gRNA-1 and 3′ gRNA-4 target sites were selected using codon optimization tool GENEisu (http://www.geneius.de/GENEius/) on human codon table. The LCLs were transfected with a plasmid using Trans-IT XP transfection reagent (Mirus Bio, Madison, WI) treated with or without a demethylating agent, 5-Aza-2′-deoxycytidine (1 μM; Abcam, Cambridge, UK), and hydralazine hydrochloride (0.2 μM; Abcam). To test whether transcripts were degraded by nonsense-mediated mRNA decay, LCL was treated by emetine (Sigma-Aldrich, St. Louis, MO) at 60 μg/ml for 12 h before RNA extraction⁵⁴. For mutation replacement gene editing (Supplementary Fig. 3E, F), LCL was co-transfected with the CAG promoter insertion plasmid and the mutation replacement genome-editing plasmid (ratio 1:3). Total RNA was extracted 48 h post-transfection using the miRNeasy plus mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A 500-ng sample of total RNA was reverse-transcribed with SuperScript IV (ThermoFisher Scientific) and oligo(dT) primers (ThermoFisher Scientific) at 55 °C for 30 min. The design of the primer sets for RT-PCR is shown in Supplementary Table 5. The RT-PCR reaction was performed with KOD One DNA polymerase (Toyobo, Osaka, Japan) at 35 cycles of 98 °C for 10 s, 60 °C for 5 s, and 68 °C for 5 s. PCR products were analyzed on agarose gels. Uncroppegel images were provided as Supplementary Fig. 5.