Puc19 plasmid

All the experiments were performed using nuclear extracts from cultured HeLa non-confluent cells (Ipracell S.A., Belgium). Benzonase was from Merck and DNase I was from Invitrogen. The magnetic streptavidin-coated beads were from Roche. The pUC 19 plasmid (Invitrogen) was used as a template for PCR-based production of biotinylated duplex DNA oligonucleotides. The PCR primers endowed with a photocleavable biotin moiety were from Eurogentec. Colloidal G250 Coomassie blue was from Thermoscientific.

Forward and reverse ssDNA target inserts were designed for Cas9 target sites. The oligos used to make the DNA targets are listed in Supplementary Table 2. The forward and reverse ssDNA sequences were annealed by heating to 95 °C for 5 mins, then cooling to 25 °C over 1 h. Next, pUC19 plasmid (Invitrogen) and the annealed dsDNA target inserts were double-digested with HindIII and XbaI (NEB). These were ligated and then transformed into DH5α E. coli. Proper insertion was confirmed by performing Sanger sequencing. DNA targets for in vitro experiments concerning HLA-C were prepared through PCR amplification of genomic DNA (gDNA) from 293 T or HeLa cells using primers listed in Supplementary Table 2.

The CVB4 E2 diabetogenic strain [9 (link)], Escherichia coli (E. coli) DH5α strain (F-φ80lacZ∆M15 ∆ (lacZYA-argF) U169 recA1 endA1 hsdR17 (r_k−, m_k+) phoA supE44 thi-1 gyrA96 relA1 λ^- Invitrogen, Waltham, MD, USA) and pUC19 plasmid (Invitrogen) [14 (link)] were used. The viral strain (kindly provided by J. W. Yoon, Julia McFarlane Diabetes Research Centre, Calgary, Alberta, AB, Canada) was propagated in the HeLa cell line from BioWhittaker in Eagle’s Minimal Essential Medium (MEM; Gibco BRL, Milano, Italy) supplemented by 10% fetal calf serum (FCS; Gibco BRL), 1% (2 mM) L-glutamine (BioWhittaker, Atlanta, GA, USA), 1% non-essential amino-acids (Gibco BRL), 50 µg/mL streptomycin, 50 IU/mL penicillin (Gibco BRL), and 0.05% (2.5 µg/mL) fungizone (Amphotericin B, Apothecon, Dabhasa, India). The supernatants were collected 3 days after inoculation, clarified at 4000× g for 10 min, divided into aliquots, and finally stored at −80 °C. Virus titer in was stocks were determined on HeLa cells by limiting dilution assay for 50% tissue culture infectious doses (TCID₅₀) using the method of Reed and Muench [15 (link)]. The bacterial strain was cultured in Luria-Bertani (LB; Invitrogen) or in LB containing 1.5% agar (Invitrogen). When required, the media was supplemented with ampicillin (Amp; 100 µg/mL).