A P P L I C AT I O N N O T E

Optimizationof reverse
transfection ofDharmacon Edit-R
synthetic crRNAand tracrRNAcomponents with
DharmaFECT
transfection
reagent in aCas9-expressing
cell line.

Introduction
Dharmacon™ Edit-R™ predesigned crRNA libraries enable
rapid, high-throughput analysis of hundreds of genes withmultiple target sites per gene. An arrayed library format permits
single-well phenotypic analysis, including high-content assays
and other morphological or reporter assays. Identification ofrelevant hits and successful screening outcomes from thesetypes of assays depend on high transfection efficiency.
To obtain the highest transfection efficiency of the Edit-R
synthetic crRNA:tracrRNA components with minimal negative
effects on cell viability, it is recommended to carefully optimize
transfection conditions for each cell line using positive control
crRNAs. A reverse transfection method can be used to increase
the throughput and reproducibility of a screen, and to moreeasily implement automated systems to carry out transfections.
Here we present an example of transfection optimizationin reverse transfection format using a recombinant U2OS
reporter cell line stably expressing Cas9 nuclease under theCAG promotor (Ubi[G76V]-EGFP-Cas9 cells). An un-cleavableubiquitin moiety (Gly76Val) fused to EGFP allows constitutive
degradation of the EGFP protein (and low basal fluorescence),
while disruption of the proteasome components by functional
protein knockout leads to accumulation of EGFP and detectable
fluorescence.

Optimization of reverse transfection of Dharmacon Edit-R synthetic crRNA and tracrRNA components with DharmaFECT transfection reagent in aCas9-expressing cell line.

Reverse transfection optimization

Results

A transfection optimization experiment should include

Transfection conditions previously determined for delivery of

two to three cell densities and a range of DharmaFECT™

siRNAs in a cell line of interest can be used as a starting pointtransfection reagent volumes. In this example, U2OS

for transfection optimization with Edit-R crRNA and tracrRNA.

Ubi[G76V]-EGFP-Cas9 cells were trypsinized, then diluted

Here we used DharmaFECT™ 4 transfection reagent thatto 5000, 10000 or 20000 cells per 80 μL in growth medium.

was previously determined to provide the highest siRNAEdit-R PPIB synthetic crRNA Control (Cat #UK-007050-01)

transfection efficiency for these U2OS cells. A range of

was used as a positive control for gene editing and Edit-R

DharmaFECT 4 transfection reagent concentrations was

crRNA targeting a proteasome gene PSMD7 was used asassessed to deliver synthetic crRNA:tracrRNA targeting

a positive control for the phenotypic assay. The crRNAs

PPIB for the highest gene editing efficiency and lowest

were co-transfected with an equimolar amount of synthetic

impact on cell viability (Figure 1). The optimal transfection

tracrRNA at a final concentration of 25 nM, using a range ofreagent concentration depended on cell density

DharmaFECT 4 transfection reagent from 0.02-0.3 μL/well.

(0.02, 0.04 or 0.12 μL/well of DharmaFECT 4 for 5000, 10000

After incubation of the crRNA:tracrRNA with the transfection

or 20000 cells per well, respectively), leading to high generegent for 20 minutes at room temperature, the transfection

editing efficiency and little effect on cell viability.

mixture was transferred to triplicate wells of tissue culture

A range of cell densities and DharmaFECT 4 transfection

plates and the suspended cells were added on top of thereagent concentrations were also assessed with synthetictransfection complex. Transfected cells were incubated in acrRNA:tracrRNA targeting PSMD7 for gene knockout using

37 °C, 5% CO2 incubator for 72 hours. A resazurin viability

the EGFP fluorescence intensity and cell viability (Figure 2).

assay and a DNA mismatch assay using T7EI endonuclease

As expected, a strong correlation was observed between

were performed 72 hours post-transfection, and EGFP

the optimal conditions for PSMD7 and PPIB crRNAs.

fluorescence was read on the Envision plate reader (Revvity)

The use of 20000 cell per well in 96-well format withfor the PSMD7 crRNA phenotypic assay control.

0.12 μL/well of DharmaFECT 4 transfection reagent resulted
in the most robust conditions with high editing efficiency(> 6 fold increase of EGFP fluorescence compared tountreated cells) as well as excellent cell viability (> 80%).

AB
0.02

DharmaFECT 4, μL/well

0.04

0.04

0.08

UTUT20000 cells/well

0.08

0.12

Viability

0.161500
850UT

120

400
200Viability, % of untreated

10000 cells/well5000 cells/well

DNA ladder
FastRuler
LR (bp)100806040200% gene editing4548 45

4240 38 48 44

29 2840 39

41 35

50

UT

0.02 0.04 0.08 0.12 0.16
5000 cells/well

UT

0.04 0.08 0.12 0.16 0.20 UT
10000 cells/well

0.08 0.12 0.16 0.20 0.30
20000 cells/wellFigure 1: Identification of optimal reverse transfection conditions using PPIB crRNA positive control. A. PPIB gene editing efficiency assessedwith DNA mismatch assay using T7EI 72 hours post-transfection (duplicate wells are shown). The amount of transfection reagent required
for successful gene editing varies with cell density. FastRuler™ Low Range DNA Ladder (Thermo Scientific, Cat #SM1103). B. Cell viability
measured with resazurin assay 72 hours post-transfection. Optimal amount of transfection reagent per well will result in cell viability of 80%or higher. Best conditions based on gene editing efficiency and viability are boxed in green. UT = untreated control, 0.02, 0.04, 0.08, 0.12, 0.16,0.20, 0.30 = μL/well of DharmaFECT 4 transfection reagent.

www.revvity.com

2

140101208100680460402020
UT0.02 0.04 0.08 0.12 0.16

UT0.04 0.08 0.12 0.16 0.20 UT 0.08 0.12 0.16 0.20 0.30

5000 cells/well

10000 cells/well
EGFP

Viability, % of untreated

EGFP, normalized to untreated

Optimization of reverse transfection of Dharmacon Edit-R synthetic crRNA and tracrRNA components with DharmaFECT transfection reagent in a
Cas9-expressing cell line.

020000 cells/well

Viability

Figure 2: Phenotypic EGFP readout of PSMD7 functional gene knockout. For all cell densities, the optimal transfection conditions determinedwith PPIB crRNA (Figure 1) correlate with the best functional gene knockout, as indicated by EGFP readout. The best conditions for each celldensity based on functional gene knockout efficiency and viability are boxed in green. UT = untreated control, 0.02, 0.04, 0.08, 0.12, 0.16,0.20, 0.30 = μL/well of DharmaFECT 4 transfection reagent.

Conclusion

Authors

Arrayed crRNA libraries allow for functional genetic screens

Elena MaksimovaŽaklina Strezoska
Melissa L. Kelley
Anja van Brabant Smithusing a wide range of phenotypic readouts, including
high-content imaging, therefore further facilitating thediscovery of genes with roles in different biological processes.

Revvity, Inc.

A reverse transfection method for delivery of arrayedsynthetic crRNA:tracrRNA libraries allows for functional geneknockout screening in an automated, high throughput manner.
Determining the optimal transfection conditions to obtain thehighest editing efficiency with minimal effects on cell viability isof utmost importance for the success of the screen.
Transfection conditions need to be optimized for every cellline keeping in mind the assay time-point and phenotypic
assay requirements, as different cell densities and growth
characteristics affect the transfection efficiency. Transfection
conditions that induce changes in cell viability and/or
provide insufficient delivery of the targeting agent canmislead researchers toward false interpretations of data,
which in the long run, are time consuming and costly.

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940 Winter Street
Waltham, MA 02451 USA
www.revvity.com

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