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  • Electroporation of Cas9 RNP (ribonucleoprotein) into adherent cells using the Neon® Electroporation

    Overview:

    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 introduction of Cas9 RNP’s into HEK293 FT cells using the Thermo Fisher Neon Electroporation System. This method uses a final concentration of 20 nM RNP per transfection in a 24-well culture plate.

    Required Materials:

    Cell Culture and Transfection

    • HEK293 cells (or other cell line) at 70-90% confluency in a T-75 flask
    • EnGen® Cas9 Nuclease NLS, S. pyogenes (M0646T or M0646M)
    • sgRNA containing the targeting sequence in the region of interest
      • sgRNAs can be generated using the EnGen sgRNA Synthesis Kit, S. pyogenes (E3322S)
      • sgRNAs must contain the target sequences (20 nucleotides) adjacent to the Protospacer Adjacent Motif (PAM, NGG) in the target DNA (1,2). See the EnGen sgRNA Synthesis Kit manual for further details.
    • ThermoFisher Neon Transfection System 10 µl Kit (MPK1025)
    • Sterile 1X PBS without Ca2+ and Mg2+
    • DMEM with Glutamax (or appropriate growth medium) with 10% FBS
    • 24-well culture plate

    DNA Extraction and Genome Editing Analysis

    • EnGen Mutation Detection Kit (E3321)
    • Epicentre QuickExtract™ DNA Extraction Solution (Epicentre #QE09050)

    Before You Start:

    Protocol:

    Electroporation

    1. Seed the cells so that they will be around 70-90% confluent on the day of transfection.
    2. Set up the RNP formation reaction as follows below:
    3. COMPONENT

      7 µl REACTION

      Resuspension Buffer R

      5.8 µl

      EnGen Cas9-NLS (20 µM)

      0.6 µl

      sgRNA (20 µM)

      0.6 µl

    4. Gently mix the reaction and incubate at room temperature for 20 minutes.
    5. During the incubation, trypsinize the cells, washing once to remove any traces of trypsin. Resuspend the cells in 10 ml of media and count.
    6. Remove 1-2 x 106 cells to a sterile microfuge tube. (One tube of cells should be enough for 10 transfections). Pellet for 5 min at 500 x g. Wash the cells once with 1X PBS and repeat the centrifugation.
    7. Resuspend the cells in 50 µl of Resuspension Buffer R.
    8. Prepare a 24-well plate by adding 500 µl growth medium to the appropriate number of wells.
    9. Add 5 µl of cells to each 7 µl RNP reaction.
    10. Aspirate 10 µl of the RNP/cells mix into a 10 µl Neon tip.  Electroporate the cells under the following conditions: 1700V, 20 ms, 1 pulse.
    11. Immediately transfer the cells to the prepared 24-well plate.
    12. Incubate the cells in a humidified 37°C, 5% CO2 incubator for 48-72 hours.

    Harvest DNA for on-target editing analysis

    1. Gently aspirate the media from the cells and wash twice with 100 µl 1X PBS. 
    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:
    3. 65°C for 15 min
      95°C for 15 min
      Hold at 4°C

    4. Dilute the DNA 1:10 in nuclease-free water
    5. Follow the protocol detailed in the EnGen Mutation Detection Kit (E3321) manual