TITLE: Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX
AUTHORS: New  England   Biolabs

[DESCRIPTION]
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.

[STEPS]
SECTION: RNP Complex Formation
1. Make a 3 Micromolar (µM) by diluting the stock with nuclease-free water.
SECTION: RNP Complex Formation
2. Make a 3 Micromolar (µM) by diluting with 1 X or Optimem.
SECTION: RNP Complex Formation
3. Form the RNP complexes as follows below:
   Component Single Reaction x3.3 (triplicates)   sgRNA (3 uM) 0.5 ul 1.65 ul   EnGen Cas9 NLS (3 uM) 0.5 ul 1.65 ul   Optimem 11.5 37.95 ul   Total 12.5 ul 41.25 ul   
SECTION: RNP Complex Formation
4. Gently mix the reaction and incubate at Room temperature for 10 min.
SECTION: RNP Complex Formation
5. Form the liposome complexes as follows below.  
   Component Single Reaction x3.3 (triplicates)   RNP (120 nM) 12.5 µl 41.25 µl   RNAiMAX 1.2 µl 3.96 µl     11.3 µl 37.29 µl     12.5 µl 82.5 µl   
 
SECTION: RNP Complex Formation
6. Gently mix the reaction and incubate at Room temperature for 20 min.
SECTION: Trypsinize and Prepare HEK293 Cells
7. Seed the cells so that they will be around 70-90% confluent on the day of transfection.
SECTION: Trypsinize and Prepare HEK293 Cells
8. During the RNP/liposome incubation, trypsinize the cells, washing once to remove any traces of trypsin.
SECTION: Trypsinize and Prepare HEK293 Cells
9. Resuspend the cells in 10 mL of media and count.
SECTION: Trypsinize and Prepare HEK293 Cells
10. Calculate the dilution and volume needed to get the cells to 3.2 x 105 cells per ml.  You will need 125 µL of cells per well.
SECTION: Transfect Cells with Liposome Complexes
11. From each tube of RNP/liposome complex, aliquot 25 µL into 3 wells of a 96-well plate.
SECTION: Transfect Cells with Liposome Complexes
12. Add 125 µL (3.2 x 105 cells/ml) to each well containing RNP/liposome complex and pipette up and down gently a few times.
SECTION: Transfect Cells with Liposome Complexes
13. Incubate the cells in a humidified 37 °C, 5% CO2 incubator for 48-72 hours.
SECTION: Harvest DNA and Amplify Target Region
14. Gently aspirate the media from the cells and wash twice with 100 µL. 
SECTION: Harvest DNA and Amplify Target Region
15. Add 75 µL and shake/vortex for 5 min. 
SECTION: Harvest DNA and Amplify Target Region
16. Transfer the solution to a PCR plate or tubes and place in a thermocycler, running the following program:
65 °C for 15 min
95 °C for 15 min
Hold at 4 °C
SECTION: Harvest DNA and Amplify Target Region
17. Dilute the DNA 1:10 in nuclease-free water.
SECTION: Harvest DNA and Amplify Target Region
18. Follow the protocol detailed in the EnGen Mutation Detection Kit (E3321S) manual.