Electroporation of EnGen® Spy Cas9 HF1 (NEB #M0667) into adherent cells using the Neon® Electroporation System


EnGen Spy Cas9 HF1 contains Simian virus 40 (SV40) T antigen nuclear localization sequence (NLS) on the N- and C- termini of the protein and may be used in vivo to create targeted genome modifications. There are several ways in which to introduce Cas9-single guide RNA complexes into cells. Here, we present a method for the introduction of Cas9 RNP complexes into HEK293 FT cells using the Thermo Fisher Neon Transfection System. This method uses a single guide RNA to protein ratio of 2:1. Actual pmols of RNA and protein used may be optimized.  

Required Materials:

Cell Culture and Transfection

  • HEK293 cells (other cell lines may need optimization) at 70-90% confluency in a T-75 flask.
  • EnGen® Spy Cas9 HF1, (NEB #M0667)
  • sgRNA containing the targeting sequence in the region of interest
    • sgRNAs can be generated using the EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322S)
    • sgRNAs must contain the target sequences (20 nucleotides) adjacent to the Protospacer Adjacent Motif (PAM, NGG) in the target DNA. See the EnGen sgRNA Synthesis Kit manual for further details.
  • Thermo Fisher Neon Transfection System 10 μl kit (MPK1025))
  • Sterile 1X PBS without Ca2+ and Mg2+
  • Trypsin to release cells
  • DMEM with Glutamax (or appropriate growth medium) with 10% FBS
  • 24-well culture plate, or desired plate

DNA Extraction and Genome Editing Analysis

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

Before You 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
  • Please refer to the Neon Transfection System manual for proper usage of the equipment.
  • The Neon 10 μl Transfection System draws 10 μl of cells and transfection material into an electroporation pipette tip.  This tip may be used twice for two sequential electroporations.  Therefore, the volumes in the protocol below allow for duplicate reactions set up in the same tube, with an overage of 5 μl.  Volumes can be adjusted according to the user’s needs.



  1. Seed the cells so that they will be around 70-90% confluent on the day of transfection.
  2. 2. Set up the RNP formation reaction as follows below. (Resuspension Buffer R is included with the Neon transfection kit.  It is not necessary to use the 10X buffer included with the EnGen Spy Cas9 HF1 for this step)

  3. Component 14.5 μl reaction
    Resuspension Buffer R 10.0 μl
    EnGen Spy Cas9 HF1 (20 uM) 2.5 μl
    sgRNA (50 uM) 2.0 μl

  4. Gently mix the Resuspension Buffer R, EnGen Spy Cas9 HF1, and gRNA 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 5-10 ml of media.  Dilute 20 μl of the cells with 20 μl of trypan blue. Determine the cell number and viability using a hemacytometer.
  6. Calculate the number of cells you will need for the entire experiment (1-2 x 105 cells per duplicate transfection) and move those to a sterile microfuge tube.  Pellet for 5 min at 500 x g.  Wash the cells once with 1X PBS and repeat the centrifugation.
  7. Calculate the volume of Resuspension Buffer R you will need to resuspend the cells (10.5 μl per transfection).  Resuspend the cells in your calculated volume.
  8. Prepare a 24-well plate by adding 500 μl growth medium to the appropriate number of wells. 
  9. Add 10.5 μl of cells to each 14.5 μl RNP reaction and pipette gently.
  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 37C, 5% CO2 incubator for 48-72 hours.

Harvest DNA and Amplify Target Region

  1. Gently aspirate the media from the cells and wash twice with 250 μl 1X PBS. 
  2. Add 50 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. Analysis of editing can be done following the protocol detailed in the EnGen Mutation Detection Kit (NEB #E3321) manual.