PROTOCOL

Enrichment of transfected cellswith Edit-R™ FluorescentCas9 Nuclease mRNA

General considerations
Edit-R Fluorescent Cas9 Nuclease mRNA enables both transfection
optimization and enrichment for gene editing experiments. Using thefluorescent mRNA for expression of both Cas9 and a fluorescent protein,either mKate2 (Evrogen, Moscow, Russia) or EGFP (University of California,San Diego), two separate proteins are translated. For enrichment
experiments, we recommend performing the transfection experiment in a
6-well tissue culture dish so that a sufficient number of cells can be harvestedfor FACS analysis according to commonly used instrument protocols. For
optimal enrichment of edited cells, we recommend sorting the cells for highmKate2 or EGFP expression levels (selecting the top 10% fluorescence) inaddition to negative and dim fluorescence for comparison. Ensure that eachfraction has enough of the total cell population such that desired fractions
can be easily expanded for downstream applications and testing.

Materials required
Edit-R CRISPR-Cas9 materials for gene engineering can be ordered athorizondiscovery.com.
• Edit-R Fluorescent Cas9 Nuclease mRNA (choose one)
1. Edit-R mKate2 Cas9 Nuclease mRNA, (1 µg/µL)
Or
2. Edit-R EGFP Cas9 Nuclease mRNA, (1 µg/µL);• Synthetic targeting guide RNA
We recommend testing at least three to five guide RNA
designs per gene of interest to identify the one withhighest editing efficiency that also results in complete
knockout of functional protein.

horizondiscovery.com• Non-targeting control guide RNA (choose one):
1. Edit-R crRNA Non-targeting Control (Cat #U-007501-XX, U-007502-XX,U-007503-XX, U-007504-XX, U-007505-XX)
Or
2. Edit-R synthetic sgRNA Non-targeting Control (Cat #U-009501-XX,U-009502-XX, U-009503-XX, U-009504-XX, U-009505-XX)• DharmaFECT™ Duo transfection reagent (1 mg/mL; Cat #T-2010-xx)

Reagents to be supplied by user
Lipid-mediated transfection and electroporation experiments require
standard cell culture reagents and instruments appropriate for maintenanceof cells. The following additional materials are required but not supplied.
•••••••••

Electroporation instrument
Electroporation reagents (buffer, cuvettes, transfer pipettes)
Flow cytometer
Multi-well tissue culture plates or tissue culture dishes
Antibiotic-free complete medium: Cell culture medium (including serumand/or supplements) recommended for maintenance and passaging of
the cells of interest without antibiotic
Serum-free medium: Cell culture medium without serum or antibiotic
but containing supplements recommended for maintenance of the cells
such as HyClone™ HyQ-RS medium (HyClone, Cat #SH30564.01)
Assay(s) for detecting gene editing events in a cell population
10 mM Tris-HCl Buffer pH 7.4 (Tris buffer), nuclease-free (Cat #B-006000-100)
Phosphate-buffered saline (PBS)General protocol for lipid transfection of Edit-R
Fluorescent Cas9 Nuclease mRNA and synthetic
guide RNAs and enrichment of transfected cells
The following is a general protocol using Edit-R Fluorescent Cas9 NucleasemRNA to enrich for transfected cells using fluorescence activated cellsorting (FACS). Exact reagent amounts and parameters for both lipidmediated transfection and electroporation should be empiricallydetermined through careful optimization in cells of interest prior toexperimentation. The protocol below describes delivery conditions inU2OS cells using the DharmaFECT Duo transfection reagent and is given
for illustrative purposes only. For further details regarding transfection
recommendations please see the full Technical Manual.

For synthetic sgRNA:a. Prepare a 2.5 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 2.5 µM.
3. In a 15 mL conical prepare for each sample to be transfected as
described in Table 2 (columns 2-4) for a final 25 nM concentration of theguide RNA and 5 µg/well of Cas9 mRNA.
4. In a separate tube, prepare a 30 μg/mL DharmaFECT Duo working
solution by diluting 30 μL of 1 mg/mL stock DharmaFECT Duotransfection reagent in 1 mL serum-free medium and mix gently; thisvolume is sufficient for 4 wells with 7.5 μL/well in 6-well format. Incubate
for 5 minutes at room temperature.

All steps of the protocol should be performed in a laminar flow cell culture
hood using sterile technique.

Table 2. Preparing transfection samples for gene editing experiments in a 6-wellplate format.

Day 1
1. Trypsinize and count cells.
2. Plate cells in 6-well plates using growth medium at a cell density sothat the cells are 70 to 90% confluent the next day. For example, U2OS
cells should be diluted to 100,000 cells in 1 mL of medium for plating at
250,000 cells/well in a 6-well plate.
3. Incubate cells at 37 °C with 5% CO2 overnight.
Table 1. Antibiotic resistances conveyed by Edit-R Lentiviral sgRNA plasmid.

Sample namePurpose

Edit-R Fluorescent Cas9 NucleasemRNA with Non-targeting
Control synthetic guide RNA

Negative control: Cas9 nuclease mRNA
without targeting RNAs

Edit-R Fluorescent Cas9 NucleasemRNA with gene-specificsynthetic guide RNA

Gene engineering sample: Cas9 nucleaseprogrammed by RNAs for targeted double-stranded break in gene of interest

Untreated

No treatment control sample: confirmationof cell viability, negative FACS sample

Day 2
1. Prepare a 100 ng/µL fluorescent Cas9 mRNA working solution bythawing fluorescent Cas9 mRNA on ice and adding 20 µL of 1 µg/µL
stock solution of fluorescent Cas9 mRNA to 180 µL of Tris buffer. Verifythe Cas9 mRNA concentration using UV spectrophotometry at 260 nm
and adjust the volume if necessary to obtain 100 ng/µL.
2. Prepare guide RNA reagents for transfection.
For crRNA and tracrRNA:a. Prepare a 10 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 10 µM.
b. Prepare a 10 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 10 µM.
c. Prepare a 2.5 μM crRNA:tracrRNA transfection complex by adding
25 μL of crRNA and 25 μL of tracrRNA to 50 μL of Tris buffer (total
volume is 100 μL).

horizondiscovery.com

Sample name

Serumfree
mediumSynthetic
guide RNAtransfectioncomplex(2.5 µM)

FluorescentCas9 mRNA
workingsolution(100 ng/µL)

DharmaFECTDuo working Growthsolution (3mediumµg/mL)

Total
volumeperwell

FluorescentCas9 NucleasemRNA withnon-targeting
control synthetic
guide RNA

175

2550250

2,0002,500

Fluorescent Cas9
nuclease mRNA
with gene-spe175
cific synthetic
guide RNA

2550250

2,0002,500

Untreated000

2,0002,500

500

Volumes (µL) are for a single well of a 6-well plate of U2OS cells to be transfected. It is
recommended to prepare sufficient sample volumes for the total number of replicates
and to account for pipetting.

5. Add 250 μL DharmaFECT Duo working solution to each sample
tube as shown in Table 2 (column 5); this will result in 3 μg/well finalconcentration. DO NOT add DharmaFECT Duo working solution to theuntransfected control, which should contain serum-free medium only.
This brings the total volume to 500 μL in each tube. Mix by pipetting
gently up and down and incubate for 20 minutes at room temperature.
6. Prepare transfection medium by adding 2,000 μL antibiotic-freecomplete medium to each sample to bring the total volume in each tube
to 2,500 μL (columns 6 and 7).
7. Remove medium from the wells of the 6-well plate containing cells andreplace with 2,500 μL of the appropriate transfection medium to each well.
Day 3
8. After 24 hours, trypsinize cells. Collect ¾ of the cells leaving ¼ as apresorted population.
9. Centrifuge and wash cell pellet with PBS to remove medium. Centrifuge
again and resuspend cells in appropriate cell sorting buffer.
Expression of fluorescent protein over time should be
examined for your cells to determine the optimal time tosort for enrichment. We suggest a minimum of 24 hours
after lipid transfection to allow for translation of themRNA into Cas9 and fluorescent proteins, and 8-24 hours
for electroporation. Cell sorting will need to be performed
before turnover of the fluorescent protein, which will bedependent on the half-life in your experimental cells.

10. Use untransfected cells as a negative sorting control to set the sorting
parameters (gate) for singlet, non-fluorescent viable cells. Sort cells into
populations based on fluorescence intensity relative to the negative
control as measured by the cell sorter instrument.
Untransfected sample
Neg
GFP-Log_Height Comp104

Top 10%

Dim103What are the maximum excitation and emission wavelengths for mKate2and EGFP?

1010100

101

102103Neg

GFP-Log_Height CompTop 10%Dim

Excitation wavelength

Emission wavelength

mKate2588 nm

633 nm

EGFP

489 nm509 nmWhat is the best way to determine optimal sorting time?
We suggest observing fluorescence intensity over time by microscopy, butalso performing a western blot for Cas9 protein or the fluorescent protein.

Transfected sample
104

Fluorescent reporter

104

mKate2 (561)-Log_Height CompIs Edit-R Fluorescent Cas9 mRNA a fusion protein?
No, the fluorescent protein and Cas9 are separate proteins translated froma single mRNA strand.

103References

102101

100

The most commonly used method for detection of editing events in acell population is a DNA mismatch detection assay (Guschin, 2010; Reyon,2012; Cong, 2013). This assay can be performed on either purified genomic
DNA or whole cell lysate using T7 Endonuclease I. When edited cells areexpanded and clonal populations are obtained, the most commonly usedmethod for characterizing gene editing is Sanger sequencing (Reyon, 2012).
See this Application Note for an example.

FAQ10210

Gene editing assay recommendations

100101102103104

mKate2 (561)-Log_Height Comp

1. L. Cong, F. A. Ran, et al. Multiplex Genome Engineering Using CRISPR/Cas
Systems. Science. 339 (6121), 819-823 (2013).
2. E. Deltcheva, K. Chylinski, et al. CRISPR RNA maturation by trans-encoded
small RNA and host factor Nuclease III. Nature. 471 (7340), 602-607 (2011).
3. D.Y. Guschin, A. J. Waite, et al. A rapid and general assay for monitoring
endogenous gene modification. Methods Mol. Biol. 649, 247-256 (2010).

Figure 1. Representative FACS data of the untransfected sample (left) andtransfected sample containing Edit-R mKate2 Cas9 Nuclease mRNA (right). The
untransfected sample should be used to define which cells are negative (Neg) for
fluorescence and to be excluded from sorting. Gates are drawn around the dim andtop 10% fluorescent populations to be sorted and collected.

For optimal enrichment, we suggest collecting cells withhigh fluorescence intensity, selecting the top 10% of
fluorescent cells.

Clonal cell lines can be created through sorting single cells
into individual wells of a 96-well plate.

11. Expand cell populations in new plates with an appropriate well size
corresponding to the number of cells collected.
12. Incubate cells at 37 °C with 5% CO2 for an additional 48 hours before
proceeding with gene editing analysis.

To find the contact information in your country for your technology of interest, please visit us at
horizondiscovery.com/contact-us
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 and EDIT-R are trademarks of Dharmacon Inc.
HYCLONE is a trademark of General Electric Company. MKATE2 is a trademark of Evrogen Inc.

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