Electroporation of Cas12a RNP (ribonucleoprotein) into adherent cells using the Lonza 4D-Nucleofector®
EnGen Lba Cas12a (Cpf1) (NEB #M0653) nuclease may be used in vivo to create targeted genome modifications. There are several ways in which to introduce Cas12a-guide RNA complexes into cells. Here we present a method for the introduction of Cas12a RNP’s into HEK293 FT cells using the Lonza 4D-Nucleofector Electroporation System. This method uses 100 pmoles of EnGen Lba Cas12a and 1000 pmoles of guide RNA per transfection in a 96-well culture plate.
Cell Culture and Transfection
HEK293 cells at 70-90% confluency in a T-75 flask
EnGen® Lba Cas12a (Cpf1) (NEB #M0653))
gRNA containing the targeting sequence in the region of interest
Lonza 4D-Nucleofector® X-Unit
SF Cell Line 4D-Nucleofector X Kit (Cat # V4XC-2032)
Sterile 1X PBS without Ca2+ and Mg2+
Trypsin to release cells
DMEM with Glutamax (or appropriate growth medium) with 10% FBS
96-well 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 hyperlink underlined text to https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination
• 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.
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:
Nucleofector Solution 7.0 µl
EnGen Lba Cas12a (Cpf1) (100 µM) 2.5 µl
gRNA (500 µM) 5.0 µl
Total reaction volume 14.5 µl
3. Gently mix the Nucleofector solution, EnGen Lba Cas12a, and gRNA and incubate at room temperature for 20 minutes.
4. 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.
5. 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.
6. 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.
7. Prepare a 96-well plate by adding 125 µl growth medium to the appropriate number of wells. Transfected samples may be plated in any number of replicates.
8. Add 10.5 µl of cells to each 14.5 µl RNP reaction.
9. 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.
10. Add 75 µl of media to the cells in the cuvette, pipetting gently. Transfer 10 µl of cells per well of the 96 well plate in the desired replicated.
11. 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 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:
• 65°C for 15 min
• 95°C for 15 min
• Hold at 4°C
3. DNA may be used undiluted or diluted. Amplification of the target region may need optimization.
4. For genome editing analysis, follow the protocol detailed in the EnGen Mutation Detection Kit (NEB #E3321) manual
BRIAN, PLEASE ADD THE FOLLOWING TO THE GENERAL TRADEMARKING PAGE. PLEASE INSERT ALPHABETICALLY IF POSSIBLE.
4D-Nucleofector® is a registered trademark of Lonza.
QuickExtract™ is a trademark of Lucigen.
Nucleocuvette™ is a trademark of Lonza.