Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX
New England Biolabs
Abstract
Cas9 nuclease may be used in vivo to create targeted genome modifications. There are several ways in which to introduce Cas9-guide RNA complexes into cells. Here we present a method for the transfection of Cas9 RNP’s into HEK293 FT cells using Thermo Fisher Lipofectamine® RNAiMAX. This is a ‘reverse transfection’ method that uses a final concentration of 10 nM RNP per transfection in a 96-well culture plate.
Before start
We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Further recommendations for avoiding ribonuclease contamination can be found here: https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination* Transfection conditions may be highly variable. It is recommended to optimize your conditions for each cell type and Cas9 target you may have. This protocol follows conditions that have been optimized for a particular target and use of HEK293 cells.
Seed the cells so that they will be around 70-90% confluent on the day of transfection. 70-90% confluent on the day of transfection.
Steps
RNP Complex Formation
Make a 3micromolar (µM) by diluting the stock with nuclease-free water.
Make a 3micromolar (µM) by diluting with 1X or Optimem.
Form the RNP complexes as follows below:
| A | B | C |
|---|---|---|
| Component | Single Reaction | x3.3 (triplicates) |
| sgRNA (3 μM) | 0.5 μl | 1.65 μl |
| EnGen Cas9 NLS (3 μM) | 0.5 μl | 1.65 μl |
| Optimem | 11.5 μl | 37.95 μl |
| Total | 12.5 μl | 41.25 μl |
Gently mix the reaction and incubate at Room temperature for 0h 10m 0s.
Form the liposome complexes as follows below.
| A | B | C |
|---|---|---|
| Component | Single Reaction | x3.3 (triplicates) |
| RNP (120 nM) | 12.5 µl | 41.25 µl |
| RNAiMAX | 1.2 µl | 3.96 µl |
| Optimem | 11.3 µl | 37.29 µl |
| Total | 12.5 µl | 82.5 µl |
Gently mix the reaction and incubate at Room temperature for 0h 20m 0s.
Trypsinize and Prepare HEK293 Cells
Seed the cells so that they will be around 70-90% confluent on the day of transfection.
During the RNP/liposome incubation, trypsinize the cells, washing once to remove any traces of trypsin.
Resuspend the cells in 10mL of media and count.
Calculate the dilution and volume needed to get the cells to 3.2 x 105 cells per ml. You will need 125µL of cells per well.
Transfect Cells with Liposome Complexes
From each tube of RNP/liposome complex, aliquot 25µL into 3 wells of a 96-well plate.
Add 125µL (3.2 x 105 cells/ml) to each well containing RNP/liposome complex and pipette up and down gently a few times.
Incubate the cells in a humidified 37°C, 5% CO2 incubator for 48-72 hours.
Harvest DNA and Amplify Target Region
Gently aspirate the media from the cells and wash twice with 100µL.
Add 75µL and shake/vortex for 0h 5m 0s.
Transfer the solution to a PCR plate or tubes and place in a thermocycler, running the following program:
65°Cfor0h 15m 0s95°Cfor0h 15m 0s- Hold at
4°C
Dilute the DNA 1:10 in nuclease-free water.
Follow the protocol detailed in the EnGen Mutation Detection Kit (E3321S) manual.
