Editing Cas9 delivery into cells

Delivery of proteins complexed with cationic lipids in cell culture Cultured cells were plated in 48-well format (250 µL volume) in Dulbecco’s Modified Eagle’s Media plus GlutaMAX (DMEM, Life Technologies) with 10% FBS (“full serum media”) and antibiotics at a cell density necessary to reach ~70% confluence the next day.

Full serum media was replaced with the same media but containing no antibiotics at least one hour before delivery.

Delivery of Cre and TALE proteins was performed by combining 1 nM to 1 µM protein (based on a 275 µL final volume) with 0.2–2.5 µL commercially available cationic lipids in 25 µL OPTIMEM media (Life Technologies) according to the manufacturer’s protocol for normal plasmid transfection, including incubation time.

25 µL of the OPTIMEM mixture containing cationic lipids and protein was then added to cells in 250 µL of full serum media.

For Cas9 delivery in vitro, purified sgRNA was incubated with Cas9 protein for 5 min before complexing with the cationic lipid reagent in 25 µL OPTIMEM and treating cells in 250 µL of full serum media.

All complexing steps were performed at room temperature. For all protein deliveries that lasted longer than 12–16 hours, media containing lipid complexes was replaced with fresh full serum media without antibiotics after 12–16 hours unless otherwise noted. Cells were assayed for recombination 48 hours after delivery, for optimal gene activation either 4 hours (TALE) or 12–16 hours (dCas9-VP64) after delivery, and for gene modification (Cas9) 48 hours after delivery.

Quantification of Cas9 protein uptake We used Alexa Fluor 647 C2 Maleimide (Life Technologies) to fluorescently label Cas9 protein on surface cysteines. A 10 mM stock solution of Alexa 647 was prepared in anhydrous DMSO (Sigma). In a 0.4 mL reaction, 10 nmol of purified Cas9 protein and 200 nmol of Alexa 647 maleimide were combined in buffer conditions used for Cas9 protein storage. The labeling reaction was incubated at 4° C for 16 hours. At the end of the reaction, excess unconjugated Alexa 647 was removed by re-purifying the labeled Cas9 protein by cation exchange chromatography as described above. To measure the amount of protein delivered into treated cells, 20,000 cells were plated in the wells of a 48-well plate 1 day prior to treatment. On the day of treatment, 50 nM of Alexa 647-labeled Cas9 (Cas9-Alexa 647) and 50 nM of EGFP sgRNA were prepared for delivery using 0.8 µL of Lipofectamine 2000 as described above, and applied to the cells. After 4 hours, Cas9-Alexa 647:sgRNA Lipofectamine-containing media was removed, and cells were washed three times with 500 µL of PBS containing 20 U/mL heparin. The cells were trypsinized and prepared for counting and flow cytometry as described above. Cas9-Alexa 647 uptake was measured by flow cytometry, while 10,000 cells of the treated population were transferred to a black, flat-bottomed, opaque 96-well plate. Standard curves of Cas9-Alexa 647 were prepared by complexing 50 pmol of the Cas9-Alexa 647 protein with Lipofectamine 2000 exactly as described for Cas9-Alexa 647 delivery, followed by serial 2-fold dilutions in DMEM with 10% FBS containing 10,000 U2OS cells per well in the 96-well plate. The effect of U2OS cells or complexation with Lipofectamine 2000 on Alexa 647 fluorescence was determined by preparing three additional Cas9-Alexa 647 standard curves: (i) with Lipofectamine 2000 in media lacking U2OS cells, (ii) without Lipofectamine 2000 in media containing U2OS cells, and (iii) without Lipofectamine 2000 in media lacking U2OS cells.
[[Category:GenomeEngineering]]