Electroporation of EnGen® Spy Cas9 HF1 (NEB #M0667) into adherent cells using the Lonza 4D-Nucleofector™ System
Overview: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 Lonza 4D-Nucleofector. This method uses a single guide RNA to protein ratio of 2:1. Actual pmols of RNA and protein used may be optimized.
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
- Lonza 4D-Nucleofector™ System
- SF Cell Line 4D-Nucleofector® X Kit
- Sterile 1X PBS without Ca2+ and Mg2+
- Trypsin to release cells
- DMEM with Glutamax (or appropriate growth medium) with 10% FBS
- Desired culture plate
DNA Extraction and Genome Editing Analysis
EnGen Mutation Detection Kit (NEB #E3321)
Epicentre QuickExtract™ DNA Extraction Solution (Epicentre #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.
- Please refer to the Lonza 4D-Nucleofector manual for proper usage of the equipment.
- The Lonza 4D-Nucleofector platform has multiple electroporation volume and vessel options. This protocol is written for use with the 16-well Nucleocuvette™ Strips.
- Seed the cells so that they will be around 70-90% confluent on the day of transfection.
- Set up the RNP formation reaction as follows below. The volumes below will allow for one (1) electroporation (with the addition of cells) of 25 μl. We have not found it necessary to account for volume overage.
- Gently mix the Nucleofector Solution, EnGen Spy Cas9 HF1, and sgRNA and incubate at room temperature for 20 minutes.
- 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 hemocytometer.
- Calculate the number of cells you will need for the entire experiment (1-2 x 105 cells per 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.
- Calculate the volume of Nucleofector Solution you will need to resuspend the cells (10.5 μl per transfection). Resuspend the cells in your calculated volume.
- Prepare a culture plate with the appropriate amount of media. Transfected samples may be plated in any number of replicates.
- Add 10.5 μl of cells to each 14.5 μl RNP reaction and pipette gently.
- Add the entire 25 µl of RNP/cell mixture to the cuvette strips. Tap lightly to ensure the liquid is on the bottom and any air bubbles have been released. Select the pre-optimized program for HEK293 cells (CM-130) and electroporate the cells.
- Add 75 µl of media to the cells in the cuvette, pipetting gently. Transfer the desired number of cells to wells in the culture plate.
- Incubate the cells in a humidified 37C, 5% CO2 incubator for 48-72 hours.
|COMPONENT||25 µl final with cells|
|Nucleofector Solution||10.0 µl|
|EnGen Spy Cas9 HF1 (20 µM)||2.5 µl|
|gRNA (50 µM)||2.0 µl|
Harvest DNA and Amplify Target Region
- Gently aspirate the media from the cells and wash twice with 250 μl 1X PBS.
- 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.
- Analysis of editing can be done following the protocol detailed in the EnGen Mutation Detection Kit (NEB #3321) manual.
65°C for 15 min
95°C for 15 min
Hold at 4°C