Sep 07, 2016

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

DOI
dx.doi.org/10.17504/protocols.io.frkbm4w
  • New England Biolabs,
  • Inc.
  • New England Biolabs (NEB)
    Tech. support phone: +1(800)632-7799 email: info@neb.com
Breton Hornblower
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.frkbm4w
External link: https://www.neb.com/protocols/2016/07/26/electroporation-of-cas9-rnp-ribonucleoprotein-into-adherent-cells-using-the-neon-electroporation
Protocol Citation: New England Biolabs, Inc. 2016. Electroporation of Cas9 RNP (ribonucleoprotein) into adherent cells using the Neon® Electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.frkbm4w
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
Created: September 03, 2016
Last Modified: March 29, 2021
Protocol Integer ID: 3596
Keywords: Cas9, gene editing, RNP, transfection, ribonucleoprotein
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 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.
Guidelines
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 (E3321S)
  • Epicentre QuickExtract™ DNA Extraction Solution (Epicentre #QE09050)
Before You Start:
Materials
MATERIALS
ReagentEnGen Cas9 NLS, S. pyogenes - 400 pmolNew England BiolabsCatalog #M0646T
ReagentEnGen sgRNA Synthesis Kit, S. pyogenes - 20 rxnsNew England BiolabsCatalog #E3322S
ReagentEnGen Mutation Detection Kit - 25 rxnsNew England BiolabsCatalog #E3321S
ReagentEpicentre QuickExtract™ DNA Extraction SolutionEpicentreCatalog #QE09050
Before start
Electroporation
Electroporation
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:
Component7 µl reaction
Resuspension Buffer R5.8 µl
EnGen Cas9-NLS (20 µM)0.6 µl
sgRNA (20 µM)0.6 µl
Gently mix the reaction and incubate at room temperature for 20 minutes.
Duration00:20:00
During the incubation, trypsinize the cells, washing once to remove any traces of trypsin.  
Resuspend the cells in 10 ml of media and count.
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.  
Duration00:05:00
Wash the cells once with 1X PBS.
Pellet for 5 min at 500 x g.  
Resuspend the cells in 50 µl of Resuspension Buffer R.
Prepare a 24-well plate by adding 500 µl growth medium to the appropriate number of wells.
Add 5 µl of cells to each 7 µl RNP reaction.
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.
Immediately transfer the cells to the prepared 24-well plate.
Incubate the cells in a humidified 37 °C, 5% C02 incubator for 48-72 hours.
Duration48:00:00
Harvest DNA for on-target editing analysis
Harvest DNA for on-target editing analysis
Gently aspirate the media from the cells.
Wash with 100 µl 1X PBS. (1/2)
Wash with 100 µl 1X PBS. (2/2)
Add 75 µl of Epicentre QuickExtract™ DNA Extraction Solution and shake/vortex for 5 minutes.
Duration00:05:00
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
Dilute the DNA 1:10 in nuclease-free water
Follow the protocol detailed in the EnGen Mutation Detection Kit (E3321S) manual