PROTOCOLElectroporation of Dharmacon™ Edit-R™
Cas9 Nuclease Expression Plasmid,
synthetic crRNA and tracrRNA for
genome engineering

Successful electroporation of the three Edit-R components (Cas9 Nuclease
Expression Plasmid, gene-specific synthetic crRNA and synthetic tracrRNA),for target gene knockout, requires careful optimization of delivery
conditions with appropriate electroporation reagents and parameters foreach cell line of interest. The protocol below assumes that experimental
conditions have been optimized previously.

Materials required
Edit-R CRISPR-Cas9 materials for gene editing can be ordered at
dharmacon.horizondiscovery.com.
• Edit-R Cas9 Nuclease Expression Plasmid with cell line-optimizedpromoter• Edit-R hCMV_mKate2-Cas9 Expression Plasmid DNA, 120 µg(Cat #U-004100-120)• Edit-R mCMV_mKate2-Cas9 Expression Plasmid DNA, 120 µg(Cat #U-004200-120)• Edit-R hEf1α_mKate2-Cas9 Expression Plasmid DNA, 120 µg (Cat #U-004300-120)• Edit-R mEf1α_mKate2-Cas9 Expression Plasmid DNA, 120 µg(Cat #U-004400-120)• Edit-R PGK_mKate2-Cas9 Expression Plasmid DNA, 120 µg (Cat #U-004500-120)• Edit-R CAG_mKate2-Cas9 Expression Plasmid DNA, 120 µg(Cat #U-004600-120)• Edit-R tracrRNA, 5, 20, 50 nmol (Cat #U-002005-XX)• Edit-R crRNA Non-targeting Control (Cat #U-007501-xx, U-007502-xx,U-007503-xx, U-007504-xx, U-007505-xx)• Edit-R crRNA, predesigned for your gene of interest in a variety of sizes,
or designed and ordered using the Dharmacon CRISPR Design Tool

It is recommended to test three to five crRNA designs pergene of interest to identify the crRNA with highest editingefficiency that also results in complete knockout offunctional protein.
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 serum
and/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 solution for resuspension of
crRNA and tracrRNA (Horizon Discovery, Cat #B-006000-100)

General protocol for electroporation of Edit-R
Cas9 Expression Plasmid and synthetic RNAs
The following example provides a general protocol using electroporation
to deliver Cas9 plasmid and crRNA:tracrRNA into cultured mammalian cells.
Electroporation of Cas9 plasmid and then sequential electroporation of
the crRNA:tracrRNA has demonstrated the best results. Exact reagents,
amounts and parameters for electroporation should be empiricallydetermined through careful optimization in cells of interest, in accordancewith electroporation instrument manufacturer’s recommendations.
The protocol below describes delivery conditions in K562 cells (2 × 106)
using the Lonza Nucleofector™ 2b instrument and is given for illustrative
purposes only. All steps of the protocol should be performed in a laminarflow cell culture hood using sterile technique.

dharmacon.horizondiscovery.com

Cell plating

9. Electroporate sample with program T-016.

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. For example,
plate 4 × 105 K562 cells in a 150 mm dish. Cell densities greater than 80%
may reduce electroporation efficiency.
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 Expression Plasmid
Negative control: Expressed Cas9with tracrRNA and Non-targeting
without targeting RNAscontrol crRNAGene editing: Expressed Cas9
Edit-R Cas9 Nuclease Expression Plasmid
programmed by RNAs for targetedwith tracrRNA and gene-specific crRNAdouble-stranded break in gene ofor positive control crRNAinterest 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 samples to be electroporated by adding 10 µg Edit-R Cas9
Nuclease Expression Plasmid to a 1.7 mL tube for each sample. TheCas9 plasmid volume to be electroporated should not exceed 10 µL(or 10% of cell resuspension volume).
6. Collect 1 × 106 cells for each sample. Centrifuge at ~ 500 × g for
5 minutes at room temperature.
7. Aspirate medium from the cell pellet and resuspend in 100 µL of Lonza
kit V electroporation buffer.
Do not leave cells resuspended in electroporation buffer for
more than 15 minutes as this can negatively affect cell viability.

It is recommended to optimize the electroporation program foryour specific cells of interest.
10. 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 back to the appropriate well.
11. Repeat steps 6-10 for remaining Cas9 plasmid samples.
12. Incubate cells at 37 °C in 5% CO2 for 30–48 hours after electroporation.
13. Prepare new 6-well plates by transferring 2 mL of pre-warmed
appropriate culture medium to each well required for the number ofsamples and replicates in the experiment. Pre-incubate/equilibrate byplacing at 37 °C in 5% CO2 while preparing samples.
14. Prepare crRNA and tracrRNA samples for electroporationa. Prepare a 100 μM tracrRNA stock solution by adding the appropriate
volume of nuclease-free, 10 mM Tris pH 7.4 buffer to tracrRNA. Verify
the RNA concentration using UV spectrophotometry at 260 nm andadjust the volume if necessary to obtain 100 µM.
b. Prepare a 100 μM crRNA stock solution by adding the appropriate
volume of nuclease-free, 10 mM Tris pH 7.4 buffer to crRNA. Verify
the RNA concentration using UV spectrophotometry at 260 nm andadjust the volume if necessary to obtain 100 µM.
c. Prepare a 50 μM working solution of crRNA:tracrRNA by combining
equal volumes of 100 µM stock solutions (1:1 ratio). Mix gently.
15. In 1.7 mL tubes prepare samples to be electroporated by adding 4 µLof 50 µM crRNA:tracrRNA working solution. This will result in 2 μM ofcrRNA:tracrRNA in the final electroporation mixture.
16. Collect cells from one well of Cas9 plasmid electroporation from step
12. Centrifuge at ~ 500 × g for 5 minutes at room temperature.
17. Aspirate medium from the cell pellet and resuspend in 100 µL of Lonza
kit V buffer.
Do not leave cells resuspended in electroporation buffer for
more than 15 minutes as this can negatively affect cell viability.
18. Transfer resuspended cells to the 1.7 mL tube containing crRNA:tracrRNAcomplex sample. Gently mix components and transfer the entire volume
to an electroporation cuvette. Sample should cover the bottom of thecuvette; tap to remove any air bubbles.
19. Electroporate sample with program T-016.
20. Use a transfer pipette to gently layer pre-incubated medium from one wellof a 6-well plate prepared in step 13, on top of electroporated cells. Gently
aspirate cells from the bottom of the cuvette and transfer to the well.
21. Repeat steps 16-20 for remaining crRNA:tracrRNA samples.
22. Incubate cells at 37 °C in 5% CO2 for a total of 48 to 72 hours after
crRNA:tracrRNA electroporation, before proceeding with geneknockout analysis.

8. Transfer resuspended cells to the 1.7 mL tube containing Cas9
plasmid. Gently mix components and transfer the entire volume toan electroporation cuvette. Sample should cover the bottom of thecuvette; tap to remove any air bubbles.

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w horizondiscovery.com/contact-us or dharmacon.horizondiscovery.com/service-and-support
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