Feb 16, 2022
Version 2
Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX V.2
- 1New England Biolabs
External link: https://www.neb.com/protocols/2016/07/26/transfection-of-cas9-rnp-ribonucleoprotein-into-adherent-cells-using-the-lipofectamine-rnaimax
Protocol Citation: New England Biolabs 2022. Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX. protocols.io https://dx.doi.org/10.17504/protocols.io.bhkuj4wwVersion created by Julia Rossmanith
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 17, 2020
Last Modified: February 16, 2022
Protocol Integer ID: 38260
Keywords: Transfection, riponucleoprotein, Cas9, Cas9 nuclease, lipofectamine,
Abstract
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 transfection of Cas9 RNP’s into HEK293 FT cells using Thermo Fisher Lipofectamine® RNAiMAX. This is a ‘reverse transfection’ method that uses a final concentration of 10 nM RNP per transfection in a 96-well culture plate.
Materials
MATERIALS
EnGen Cas9 NLS, S. pyogenes - 400 pmolNew England BiolabsCatalog #M0646T
EnGen sgRNA Synthesis Kit, S. pyogenes - 20 rxnsNew England BiolabsCatalog #E3322S
EnGen Mutation Detection Kit - 25 rxnsNew England BiolabsCatalog #E3321S
Epicentre QuickExtract™ DNA Extraction SolutionEpicentreCatalog #QE09050
Opti-MEM™ Reduced Serum MediumThermo Fisher ScientificCatalog #31985062
HEK293ATCCCatalog #CRL-1573
DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
Lipofectamine™ RNAiMAX Transfection ReagentThermo FisherCatalog #13778030
DMEM, low glucose, GlutaMAX™ Supplement, pyruvateThermo FisherCatalog #21885108
Required Materials:
Cell Culture and Transfection
- HEK293 cells (or other cell line) at 70-90% confluency in a T-75 flask.
- sgRNA containing the targeting sequence in the region of interest
- 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.
- Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher)
- Sterile 1X PBS without Ca2+ and Mg2+
- DMEM with Glutamax (or appropriate growth medium) with 10% FBS
- Optimem Reduced Serum Medium (ThermoFisher)
- 96-well culture plate
DNA Extraction and Genome Editing Analysis
- Epicentre QuickExtract™ DNA Extraction Solution (Epicentre #QE09050)
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before 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
- 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.
Seed the cells so that they will be around 70-90% confluent on the day of transfection.
RNP Complex Formation
RNP Complex Formation
Make a 3 micromolar (µM) working solution of sgRNA by diluting the stock with nuclease-free water.
Make a 3 micromolar (µM) working solution of Cas9-NLS by diluting with 1 X Cas9 Reaction Buffer or Optimem.
Form the RNP complexes as follows below:
| A | B | C | |
| Component | Single Reaction | x3.3 (triplicates) | |
| sgRNA (3 μM) | 0.5 μl | 1.65 μl | |
| EnGen Cas9 NLS (3 μM) | 0.5 μl | 1.65 μl | |
| Optimem | 11.5 μl | 37.95 μl | |
| Total | 12.5 μl | 41.25 μl |
Gently mix the reaction and incubate at Room temperature for 00:10:00 .
Form the liposome complexes as follows below.
| A | B | C | |
| Component | Single Reaction | x3.3 (triplicates) | |
| RNP (120 nM) | 12.5 µl | 41.25 µl | |
| RNAiMAX | 1.2 µl | 3.96 µl | |
| Optimem | 11.3 µl | 37.29 µl | |
| Total | 12.5 µl | 82.5 µl |
Note
You can make a master mix of the RNAiMAX and Optimem and add this directly to the RNP tube from above.
Gently mix the reaction and incubate at Room temperature for 00:20:00 .
Trypsinize and Prepare HEK293 Cells
Trypsinize and Prepare HEK293 Cells
Seed the cells so that they will be around 70-90% confluent on the day of transfection.
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.
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
Transfect Cells with Liposome Complexes
From each tube of RNP/liposome complex, aliquot 25 µL into 3 wells of a 96-well plate.
Add 125 µL cells (3.2 x 105 cells/ml) to each well containing RNP/liposome complex and pipette up and down gently a few times.
Incubate the cells in a humidified 37 °C , 5% CO2 incubator for 48-72 hours.
Harvest DNA and Amplify Target Region
Harvest DNA and Amplify Target Region
Gently aspirate the media from the cells and wash twice with 100 µL 1X PBS .
Add 75 µL Epicentre QuickExtract™ DNA Extraction Solution and shake/vortex for 00:05:00 .
Transfer the solution to a PCR plate or tubes and place in a thermocycler, running the following program:
- 65 °C for 00:15:00
- 95 °C for 00:15:00
- Hold at 4 °C
Dilute the DNA 1:10 in nuclease-free water.