Example 2
The lentivirus construct used to generate Cas9-expressing T cells contains either the SFFV or the EF1a promoter expressing spCas9-T2A followed by a blasticidin or Thy1.1 selection cassette. To generate lentivirus containing Cas9, 289×106 of LentiX-293 T cells were plated out in a 5-layer CellSTACK 24 hours prior to transfection. 18 mL of serum-free OptiMEM and 1212 μL of TranslT-293 were combined and incubated for 5 minutes before combining helper plasmids (58 μg VSVG and 115 μg PAX2-Gag-Pol) with 231 μg of plasmid encoding Cas9 and either blasticidin or Thy1.1. After 20 minutes, this mixture was added back to cells with fresh media. Media was replaced 18 hours after transfection, and virus was collected 48 hours post-transfection. After passing through a 0.45 μm filter, the virus was concentrated with an Amicon spin filtration column so that final titer of infectious units was 10×106 infectious units per mL, as determined by function titration on HT1080 cells. Virus was aliquoted and stored at −80° C.
Cell lines were infected with Cas9-expressing lentivirus (pKSQ006) and selected with blasticidin S for at least one week. Expression of Cas9 protein was verified by immunofluorescence and flow cytometry with an anti-Cas9 antibody (Cell Signaling Technologies).