PROTOCOLElectroporation of Edit-R™ Cas9 nuclease
protein NLS and synthetic guideRNA for genome engineering

Successful electroporation of Cas9 Nuclease protein with Nuclear
Localization Signal (NLS), and synthetic guide RNA [single guide RNA(sgRNA) or crRNA complexed with tracrRNA] with subsequent geneknockout, requires careful optimization of delivery conditions withappropriate electroporation reagents and parameters for each cell line ofinterest. The protocol below assumes that experimental conditions havebeen optimized previously.

• Synthetic targeting guide RNA (choose one):
1. Edit-R synthetic sgRNAor
2. Edit-R synthetic crRNA and tracrRNA oligos
We recommend testing three to five guide RNA designs
per gene of interest to identify the guide RNA with highest
editing efficiency that also results in complete knockout offunctional protein.

Materials required
Edit-R CRISPR-Cas9 materials for gene editing can be ordered athorizondiscovery.com.

• Non-targeting control guide RNA (choose one):
1. Edit-R synthetic sgRNA Non-targeting Control (Cat #U-009501-XX,U-009502-XX, U-009503-XX, U-009504-XX, U-009505-XX)• Edit-R Cas9 Nuclease protein NLS
Concentration

Quantity

Catalog #µMµg/µLpmolµg

CAS11728*

203.225040

CAS11200*

203.250080

CAS11201*203.21000

160

CAS11729*

406.450080

CAS11730*

406.42 × 500

160

CAS12205

61.8

10618

100

CAS12206

61.8

103090500

CAS12207

61.8

105 × 3090

5 × 500* Discontinued.

horizondiscovery.com

or
2. Edit-R crRNA Non-targeting Control (Cat #U-007501-XX, U-007502-XX,U-007503-XX, U-007504-XX, U-007505-XX)
Electroporation experiments require standard cell culture reagents andinstruments appropriate for maintenance of cells. The following additional
materials are required but not supplied:••••Electroporation instrument
Electroporation reagents (buffer, cuvettes, transfer pipettes)
Multi-well tissue culture plates or tissue culture dishes
Antibiotic-free complete medium: Cell culture medium (including serumand/or supplements) without antibiotic; recommended for maintenanceand passaging of the cells of interest• Assay(s) for detecting gene editing events in a cell population• 10 mM Tris pH 7.4 nuclease-free buffer (Tris buffer) solution(Dharmacon, Cat #B-006000-100)

General protocol for electroporation of Edit-R
Cas9 Nuclease protein NLS and synthetic RNAs
The following example provides a general protocol using electroporation
to deliver Cas9 protein and guide RNA into cultured mammalian cells.
Exact reagents, amounts and parameters for electroporation shouldbe empirically determined through careful optimization in cells ofinterest, in accordance with electroporation instrument manufacturer’srecommendations. The protocol below describes delivery conditions inU2OS cells (1 × 106) using the Lonza Nucleofector™ 2b instrument and isgiven for illustrative purposes only. All steps of the protocol should be
performed in a laminar flow cell culture hood using sterile technique.

Cell plating
Optimal cell number for plating will vary with growth characteristics ofspecific cells and should be determined empirically.
1. Trypsinize and count cells.
2. Plate cells to achieve 70–80% confluence the next day. Cell densities
greater than 80% may reduce electroporation efficiency. For example,plate 2.5–3 × 106 U2OS cells in a 150 mm dish.
3. Incubate cells at 37 °C in 5% CO2 overnight.

Electroporation
Table 1. Recommended samples for a gene editing electroporation experiment.

SampleExplanation of sample

Edit-R Cas9 Nuclease protein NLS
with Non-targeting control guide RNA

Negative control: Cas9 protein
without targeting guide RNAs

Edit-R Cas9 Nuclease protein NLS withgene-specific guide RNA (and tracrRNA
if using crRNA for guide format)

Gene editing: Cas9 protein
programmed by guide RNAs fortargeted double-stranded break ingene of interest or control gene

Untransfected

No treatment control:
confirmation of cell viability

It is recommended to perform electroporation of gene-specificguide RNA in triplicate along with the controls listed in Table 1
for high confidence experimental results.
4. Prepare 6-well plates by transferring 2 mL of pre-warmed culture
medium to each well required for the number of samples and replicatesin the experiment. Pre-incubate/equilibrate by placing at 37 °C in 5% CO2
while preparing samples.
5. Prepare synthetic sgRNA or crRNA:tracrRNA samples for electroporation.
For synthetic sgRNAa.

Prepare a 100 μM synthetic sgRNA stock solution by adding theappropriate volume of Tris buffer to the sgRNA. Verify the RNA
concentration using UV spectrophotometry at 260 nm and adjust thevolume if necessary to obtain 100 μM.

For crRNA and tracrRNAa. Prepare a 200 μM crRNA stock solution by adding the appropriate
volume of Tris buffer to crRNA. Verify the RNA concentration using UV
spectrophotometry at 260 nm and adjust the volume if necessary toobtain 200 μM.
b. Prepare a 200 μM tracrRNA stock solution by adding the appropriate
volume of Tris buffer to tracrRNA. Verify the RNA concentration using
UV spectrophotometry at 260 nm and adjust the volume if necessary
to obtain 200 μM.
c. Prepare a 100 μM working solution of crRNA:tracrRNA by combining
equal volumes of 200 μM stock solutions (1:1 ratio). Mix gently.
6. In 1.7 mL tubes, prepare samples to be electroporated. Combine
150 pmol of Cas9 protein with 3 µL (300 pmol) of crRNA:tracrRNA orsynthetic sgRNA working solution for a final concentration of 1.5 µM
and 3 µM, respectively. This creates the Cas9 ribonucleoprotein(RNP) complex.
Concentration

150 pmol

Total volume20 µM7.4 µL

10.4 µL

40 µM

3.7 µL6.7 µL61.8 µM

2.4 µL

5.4 µL

Lower amounts of Cas9 protein with higher amounts ofguide RNA can be electroporated. We found that using 100pmol of Cas9 protein with 400-600 pmol guide RNA produced comparable gene editing as 150 pmol Cas9 proteinwith 300 pmol guide RNA; this ratio should be empiricallydetermined for your cell line.

7. Incubate at room temperature for 10–15 minutes.
8. Collect 1 × 106 cells for each sample. Centrifuge at ~ 500 × g for 1 minuteat room temperature.
9. Aspirate medium from the cell pellet and resuspend in 100 µL ofappropriate electroporation buffer.
Do not leave cells resuspended in electroporation buffer for
more than 15 minutes as this can negatively affect cell viability.

10. Transfer resuspended cells to the 1.7 mL tube containing Cas9 RNPcomplex. Gently mix components and transfer the entire volume toan electroporation cuvette. Sample should cover the bottom of thecuvette; tap to remove any air bubbles.
11. Electroporate sample with program X-001.
12. Use a transfer pipette to gently layer pre-incubated medium on top ofelectroporated cells from one well of a 6-well plate. Gently aspirate cells
from the bottom of the cuvette and transfer to the well.
13. Repeat steps 8–12 for remaining Cas9 protein gene editing samples.
14. Incubate cells at 37 °C in 5% CO2 for a total of 48 to 72 hours after
electroporation before proceeding with gene knockout analysis.

For more information

Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom©2020 The Horizon logo and other trademarks are the property of Horizon Discovery Limited, unless otherwise stated.
DHARMACON is a trademark of Dharmacon Inc. Nucleofector is a trademark of Lonza Group.

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