CRISPR/Cas9 on Raji WT cells in presence of cell-penetrating peptide
The human GeCKO v2 library (2 plasmid system) (Addgene, ref. no. 1000000049) was amplified by electroporation using the electroporation system (Bio-Rad Gene Pulser II #165-2105) and the Lucigen Endura cells (ref. no. 60242). Cells were plated on LB Agar plate containing 100 g/mL ampicillin. After 14 hours at 32C, colonies were scrapped and plasmids recovered with the Plasmid Maxi kit (Qiagen, ref. no. 12162). To produced lentivirus library, 12 T-225 flasks were seeded with 12x106 of HEK293T cells per flask. The day after, each flask was treated as follows: 10 g pMD2.G, 30 g psPAX2 and 25 g GeCKO plasmid library were mixed with 250 mM CaCl2. This solution was mixed (v/v) with 2x HEPES buffer (NaCl 280 mM, KCl 10 mM, Na2HPO4 1.5 mM, D-glucose 12 mM, HEPES 50mM), incubated for one minute and added to the culture medium. After seven hours, the medium was removed and replaced by DMEM supplemented with 10 % FBS and one percent penicillin streptomycin. After 48 hours, the medium was collected and centrifuged 5 min at 2000xg to pellet the cells. Supernatant was filtered through a 0.45 m HV/PVDF (Millipore, ref. no. SE1M003M00) and concentrated 100 times by ultracentrifugation (Beckman) at 24.000 rpm for two hours at 4C. Virus pellet was resuspended in ice-cold PBS, aliquoted and stored at -80C. To express the Cas9 endonuclease, Raji cells were infected with lentiCas9-Blast (Addgene, ref. no. 52962) and selected with 10 g/mL blasticidin for a week. The multiplicity of infection (MOI) of the GeCKO virus library was determined as follow. Different volumes of virus library were added to 3x106 Raji cells expressing Cas9 plated in a 12-well plate. After 24 hours, each well was split into duplicate and one replicate received 10 g/mL puromycin for 3 days. Cell viability was determined by trypan blue exclusion and MOI was considered as the percentage of living cells. The virus volume yielding to MOI close to 0.3 was chosen. Large-scale infection of 12x107 Raji cells was carried out in 12-well plates with 3x106 cells per well. After 24 hours, wells were pooled in a T-225 flask and infected cells selected with 10 g/mL puromycin for a week. Thirty million infected untreated Raji cells were frozen for genomic DNA analysis. Sixty million infected Raji cells were treated with 40 M TAT-RasGAP317-326 for 8 days with a medium and peptide renewal every 2-3 days. Thirty million Raji cells, selected in presence of TAT- RasGAP317-326, were frozen for genomic DNA analysis. Similar selection was performed with SKW6.4 cells during 17 days. Genomic DNA was extracted with Blood & Cell Culture DNA Midi Kit according to manufacturers instructions (Qiagen, ref. no. 13343). A first PCR was performed to amplify the lentiCRISPR sgRNA region with the following primers. A second PCR was performed to attach Illumina adaptors and barcodes to samples. Both PCR were performed in 100 L with the Herculase II Fusion DNA Polymerase (Agilent, ref. no. 600675). Amplicons were gel extracted, quantified, mixed and sequenced with a MiSeq (Illumina). Raw FASTQ files were demultiplexed and processed to contain only the unique sgRNA sequence. The number of reads of each sgRNA was normalized as described 58. The MAGeCK algorithm 59 was used to rank screening hits by the consistent enrichment among multiple sgRNAs targeting the same gene.
SUB4501054
Raji_WT_CPP