Arrayed knockout generation with Cas9-RNPs
For Caco-2 transfection, 10 pmol Streptococcus Pyogenes NLS-Sp.Cas9-NLS (SpCas9) nuclease (Aldevron; 9212) was combined with 30 pmol total synthetic sgRNA (10 pmol each sgRNA, Synthego) to form ribonucleoproteins (RNPs) in 20 µl total volume with SF Buffer (Lonza V5SC-2002) and allowed to complex at room temperature for 10 min. All cells were dissociated into single cells using TrypLE Express (Gibco), resuspended in culture media and counted. 100,000 cells per nucleofection reaction were pelleted by centrifugation at 200 × g for 5 min. After centrifugation, cells were resuspended in transfection buffer according to cell type and diluted to 2 × 10^4 cells/ml. Five µl of cell solution was added to preformed RNP solution and gently mixed. Nucleofections were performed on a Lonza HT 384-well nucleofector system (Lonza, #AAU- 1001) using program CM-150 for Caco-2. Immediately after nucleofection, each reaction was transferred to a tissue-culture treated 96- well plate containing 100 µl of normal culture media and seeded at a density of 50,000 cells per well. Transfected cells were incubated following standard protocols.