Edit r system

Capan-2, MCF7 and T3M4 were respectively seeded into 96-wells plates at a density of 8 × 10³, 1 × 10⁴ and 2 × 10⁵ cells per well in triplicate and were maintained in complete medium to reach 70% of confluence on the next day. Cells were incubated at 37 °C in a 5% CO₂ humidified atmosphere overnight. Transfection of RNP complex into Capan-2, MCF7 and T3M4 cells was next performed following the protocol entitled «Transfection of ssDNA donor oligonucleotides for HDR-mediated gene modifications using the Dharmacon™ Edit-R™ system» and using a lipid-based transfection reagent called DharmaFECT Duo (Dharmacon, Cat #T-2010-01). Briefly, a 2.5 µM Cas9 protein working solution, a 1 µM donor oligo working solution, a 2 µM gRNA transfection complex (with crRNA:tracrRNA) and a 6 µg/mL DharmaFECT Duo working solution were prepared from the stock solutions. RNP complex was then assembled with Cas9 protein at 25 nM, donor oligo at 10 nM and gRNA at 50 nM. The final transfection mixture was consisted of RNP complex, transfection reagent and serum-free medium, and was incubated 18 h under usual culture conditions. Two controls were also prepared: a gene editing control (RNP complex without donor oligo, CRISPR/Cas9 control (CC_ctrl)) and a negative control (non-transfected cells).

MPI gene was knocked out by CRISPR–Cas9 gene editing using the Edit-R system (Dharmacon), in accordance with the manufacturer’s instructions. Briefly, HT1080 cells or HeLa cells were seeded and cultured for 24 hr prior to transfection. Synthetic CRISPR RNAs (CM-011729-01 and CM-011729-03, Dharmacon; 10 μM, 2 μL each) targeting the MPI gene were mixed with 4 μL of 10 μM trans-activating CRISPR RNA (tracrRNA; U-002005-05, Dharmacon) and transfected with 1 μg of Edit-R SMART Cas9_mCMV_(PuroR) expression plasmid (U-005200-120, Dharmacon) using 5 μL of Lipofectamine 2000 (11668027, Thermo Fisher Scientific) in 400 μL of Opti-MEM (31985062, Thermo Fisher Scientific). Transfectants were selected in complete medium supplemented with 50 μM Man and 1 μg/mL puromycin, and then cloned by limiting dilution. For HT1080 cells, MPI-KO clones were screened by polymerase chain reaction (PCR) using genomic DNA as a template and two primer sets (Supplementary file 1) for amplifying the wild-type allele and for the KO allele of the MPI locus. The PCR products for the KO allele were subjected to Sanger sequencing. For HeLa cells, MPI-KO clones were screened by mannose auxotrophy and sensitivity, as well as western blotting using anti-MPI antibody (see below).