Overview:

Cas9 nuclease and its variants should be used in vitro and in vivo to create RNPs. 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 HEK293T cells using Thermo Fisher Lipofectamine^® RNAiMAX. This is one of the methods that use a final concentration of 10 nM RNP per transfection in a 96-well culture plate.

Materials required:

Cell Culture and Transfection

1) HEK293 cells (or other adherent cell line) at 70-90% confluency in a T-75 flask

2) Cas9 Nuclease with NLS, S. pyogenes

3) sgRNA containing the targeting sequence in the region of interest

· sgRNAs can be generated using the sgRNA Synthesis Kit, S. pyogenes of NEB

· sgRNAs must contain the target sequences (20 nucleotides) adjacent to the Protospacer Adjacent Motif (PAM, NGG) in the target DNA

4) Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher)

5) Sterile 1X PBS without Ca2+ and Mg2+

6) Appropriate growth medium with FBS

7) Opti-MEM Reduced Serum Medium (ThermoFisher)

8) 96-well culture plate

 

Attention:

Strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase 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

Protocol:

RNP Complex Formation

1.  1. Make a 3 µM working solution of sgRNA by diluting the stock with nuclease-free water.

2.   2. Make a 3 µM working solution of Cas9-NLS by diluting with 1X Cas9 Reaction Buffer or Opti-MEM. 

3.    3. Form the RNP complexes as follows below:

   4. Gently mix the reaction and incubate at room temperature for 10 minutes. 

2.   5. Form the liposome complexes as follows below.  You can make a master mix of the RNAiMAX and Opti-MEM and add this directly to the RNP tube from above.

    6.Gently mix the reaction and incubate at room temperature for 20 minutes.

Trypsinize and Prepare HEK293 Cells

1.     1.Seed the cells so that they will be around 70-90% confluent on the day of transfection.

2.     2. During the RNP/liposome incubation, trypsinize the cells, washing once to remove any traces of trypsin. Resuspend the cells in 10 ml of media and count.

3.   3. 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.

Transfect Cells with Liposome Complexes

1.     1. From each tube of RNP/liposome complex, aliquot 30 µl into 3 wells (10 µl/well ) of a 96-well plate.

2.  2. Add 125 µl of cells (3.2 x 10⁵ cells/ml) to each well containing RNP/liposome complex and pipette up and down gently a few times.

3.   3. Incubate the cells in a humidified 37°C, 5% CO₂ incubator for 48-72 hours.

Harvest DNA and Amplify Target Region

1.  1. Gently aspirate the media from the cells and wash twice with 100 μl 1X PBS. 

2. 2. Add 75 µl of Epicentre QuickExtract DNA Extraction Solution and shake/vortex for 5 minutes. 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

3.  3. Dilute the DNA 1:10 in nuclease-free water.

 

4. 4. Follow the protocol detailed in NEB’s EnGen Mutation Detection Kit (E3321) manual.