PROTOCOL

Reverse transfection of arrayed crRNAThis protocol is for reverse transfection of Dharmacon Edit-R crRNA arrayed libraries.

Table of contents
1. Reverse transfection of arrayed crRNAs..............................1
Product description.................................................................................. 1
Materials........................................................................................................ 1
Guidelines for resuspension of the crRNA....................................... 2
Reverse transfection of arrayed crRNA.............................................. 2
2. Appendix.................................................................................3
Optimization of transfection conditions for delivery of
crRNA:tracrRNA.......................................................................................... 3
Gene editing assay recommendations.............................................. 3
3. Frequently asked questions..................................................3

1 Reverse transfection of arrayed crRNAs
Product description
Libraries of predesigned synthetic crRNAs provide an opportunity to apply
the CRISPR-Cas9 system for functional gene knockout analysis in an arrayed
format. Dharmacon™ Edit-R™ crRNA libraries consist of synthetic crRNAs thatare predesigned using the proprietary Edit-R CRISPR RNA algorithm. Thisalgorithm was trained on functional knockout data and has demonstrated
the ability to select guide RNA target regions more likely to give functional
knockout of the protein, not just create a DNA cut. The algorithm
additionally includes specificity scoring using an internal alignment tool forcomplete off-target identification, identifying both mismatches and gaps.
Edit-R crRNA Libraries are available as the following:• Catalog libraries of predefined gene family collections for humanand mouse»» Four crRNA per gene, 80 wells per plate, columns 1 and 12 left empty,
0.5 nmol per well in 96-well plates»» Provided in NUNC Polystyrene 96-well V-bottom plates (Cat #249952)• Cherry-pick crRNA libraries are created by a researcher from their own

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gene list. Learn more or get started at dharmacon.horizondiscovery.
com/rnai/screening-libraries/cherry-pick-libraries»» One to five crRNA per gene (minimum of three is recommended) in
96-well plates, minimum 40 wells»» Customizable plate layout; Edit-R catalog control crRNAs may be
added to any wells within the plate(s).
»» Provided in NUNC Polystyrene 96-well V-bottom plates (Cat #249952)

Materials• Plates of synthetic crRNAs, up to 2 nmol per well in 96-well plates
Additional required materials are listed below and are not provided with
crRNA library purchase:
Edit-R tracrRNA, 5, 20, 50, 200, 500 nmol, 1 µmol and 5 x 1 µmol 		(Dharmacon, Cat #U-002005-50/200/500/1000/5000).
tracrRNA is required for use with all synthetic crRNA reagents.
• DharmaFECT transfection reagent (formulation is dependent on
specific cell line of interest)»» DharmaFECT 1 Cat #T-2001-XX»» DharmaFECT 2 Cat #T-2002-XX»» DharmaFECT 3 Cat #T-2003-XX»» DharmaFECT 4 Cat #T-2004-XX• 10 mM Tris pH 7.4 nuclease-free buffer solution (Dharmacon,
Cat #B-006000-100)• 96-well tissue culture plates• 96-well V-bottom polystyrene standard storage plates or deep wellplates (for example, NUNC Cat #249952 or Cat #12-565-553)• Assay for cell viability such as CellTiter-Blue® Cell Viability Assay(Promega Corp., Cat #G8081)• Assay(s) for detecting gene editing events in a cell population(https://dharmacon.horizondiscovery.com/uploadedFiles/Resources/
crrna-positive-controls-protocol.pdf)• Positive control crRNA and detection primers for assessment ofgene editing (https://dharmacon.horizondiscovery.com/gene-editing/crispr-cas9/crispr-controls/edit-r-synthetic-positive-crrna-controls/)• Non-targeting control crRNA (https://dharmacon.horizondiscovery.
com/gene-editing/crispr-cas9/crispr-controls/edit-r-synthetic-crrnanon-targeting-controls/)• Assay-specific positive control crRNA (defined by researcher)• Growth medium: antibiotic-free cell culture medium (with serum and/or supplements) recommended for maintenance of the cells of interest• Serum-free and antibiotic-free cell culture medium for preparation oftransfection mix (for example, MEM-RS, HyClone Cat # SH30564).

•
•
•

Day 1
This protocol is for transfection of one arrayed crRNA 96-well plate, in
triplicate, at final 25 nM concentration of the crRNA:tracrRNA. Calculations
are done for quadruplicates providing excess for the ease of pipetting.

For phenotypic analysis with the arrayed synthetic crRNA libraries
we strongly recommend using cell lines that constitutively express
Cas9 nuclease. Transfection of crRNA:tracrRNA into a cell line that isconstitutively expressing Cas9 nuclease results in a higher percentage
of edited cells thus allowing for easier downstream high-throughput
phenotypic analysis. For generation of the Cas9 stable expressing
cells please follow recommendations in the Gene Engineering with
Lentiviral Cas9 Particles and Synthetic CRISPR RNAs manual.

1.

The phenotypic analysis, including assay optimization
and analysis, is cell line and assay specific and requires
optimization by the researcher.

2.

Guidelines for resuspension of the crRNA
1. The crRNA libraries are shipped at ambient temperature as dry pellets
of RNA in each well and should be stored at -20 °C upon arrival in amanual defrost or non-cycling freezer. If necessary, crRNAs can be
stored as dry pellets (unopened) at 4 °C for several weeks.
2. Briefly centrifuge plates to ensure that the crRNA is collected at thebottom of the well.
3. Wipe adhesive foil cover with 70% ethanol or other RNasedecontamination solution.
4. Pierce or carefully peel back the foil seal to gain access to wells. Usecaution and avoid shredding the seal.
5. If you are starting with a plate of 0.5 nmol per well, resuspend arrayed
crRNAs to 10 μM solution by adding 50 μL nuclease-free 10 mM Tris
pH 7.4 buffer to 0.5 nmol of crRNA (for different quantities of crRNAs
see Table 3).
6. Pipette solution up and down 3-5 times while avoiding introductionof bubbles.
7. Seal the plate and place it on an orbital mixer/shaker for 70-90
minutes at room temperature.
8. Briefly centrifuge plates to collect solution to bottom of the wells.
9. From this master crRNA plate, generate plates with 1 μM
working concentration of crRNAs using nuclease-free, 10 mM Tris pH
7.4 buffer. This eliminates the subsequent requirement for pipettingof very small volumes.
10. crRNA plates may now be used immediately, aliquoted into single-use
plates or stored at -20 °C in a manual defrost or non-cycling freezer.
11. For storage, seal plates with appropriate adhesive or heat seals.

Reverse transfection of arrayed crRNAThe following is a general protocol for reverse transfection of arrayed
crRNA libraries using stable Cas9-expressing mammalian cells. For
guidelines using arrayed crRNA plates with forward transfection see
https://dharmacon.horizondiscovery.com/uploadedFiles/Resources/editr-crrna-libraries-guide-protocol.pdf. Optimal plating density will dependon growth characteristics of specific cell lines and assay requirements
and these parameters should be determined experimentally. Exact
parameters for crRNA:tracrRNA transfection in your cells of interest
should be empirically determined through careful optimization prior
to experimentation see Appendix for Optimization of transfection
conditions with crRNA:tracrRNA. Catalog crRNA library plates are suppliedwith columns 1 and 12 empty to allow addition of researcher-defined
controls. We suggest including the following controls:

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Untreated cells
Positive control crRNANegative control: non-targeting crRNATransfer 10 μL of 1 μM working crRNA solution to each well of a
96-well V-bottom transfection mixture plate (NUNC polystyrene
96 well V- bottom plates Cat #249952 or other appropriate plates
may be used).
Positive and negative crRNA controls can be added to empty
wells of the V-bottom transfection mixture plate (columns 1
and 12 in catalog libraries).

Resuspend Edit-R tracrRNA to 10 μM stock solution in nuclease-free,
10 mM Tris pH 7.4 buffer (for example, add 500 μL to 5 nmol of
tracrRNA).
3. Prepare 333 nM tracrRNA working solution by adding 120 µL of
10 μM tracrRNA stock solution to serum-free medium for a total
volume of 3.6 mL. This will allow preparation of one arrayed crRNAlibrary plate in triplicate (total of three plates) and includes excess forease of pipetting.
4. Add 30 µL of 333 nM working tracrRNA solution to each well of
96-well V-bottom transfection mixture plate containing 10 µL of
crRNA, prepared in step 1. This will bring the concentration of the
crRNA and tracrRNA to 250 nM.
5. Prepare transfection reagent working solution by diluting thetransfection reagent stock solution in serum-free medium for a total
volume of 5 mL. This volume will allow preparation of one crRNAlibrary plate in triplicate and includes excess for ease of pipetting.
For example, if the optimal amount of transfection reagent wasdetermined to be 0.05 µL per well of cells, add 25 µL of transfection
reagent stock solution to serum-free medium for a total volume of
5 mL. For preparations of other transfection reagent concentrations
see Table 1.
6. Add 40 μL of transfection reagent working solution to each wellof 96-well V-bottom transfection mixture plate containing thecrRNA:tracrRNA complex. This brings the total volume to 80 μL.
7. Immediately mix by pipetting gently up and down and incubate for
20 minutes at room temperature.
8. During this incubation time, cells may be prepared for seeding.
Trypsinize cells, spin down, remove medium and resuspend in cell
growth medium such that when 80 μL are added per well it will resultin the previously determined optimal cell density for your transfection.
For example, if optimal cell density was determined to be 10,000 cells
per well, prepare 28 mL of cell suspension at 1.25 × 105 cells/mL forthree plates.
9. Briefly mix (by pipetting) the transfection mix prepared in step 7
and add 20 μL of the transfection mixture to each well of three 96-welltissue culture plates.
10. Add 80 μL of the cell suspension prepared in step 8 to each well of
the three 96-well tissue culture plates. This will bring the volume to
100 μL and the final concentration of the crRNA:tracrRNA complex
to 25 nM.
11. Incubate transfected cells at 37 °C in a humidified CO2 incubator for
48-72 hours before proceeding with the phenotypic assay or gene
editing analysis.

2conditions that have previously been optimized for siRNA delivery are areasonable starting point for crRNA:tracrRNA transfection optimization.
Cell type-specific guidelines for the four DharmaFECT transfection reagent
formulations can be found in the DharmaFECT Cell Type Guide.

Table 1. Transfection reagent amounts for transfection of one 96-well crRNA libraryplate in triplicate.

Transfection reagent
volume per wellof cells (μL)

Transfection reagent
volume (μL)

Serum-free medium
volume (mL)

0.01

5

5.00

0.025

12.5

4.99

0.05

25

4.98

0.1

50

4.95

0.2

100

4.90

The transfection reagent amounts for reverse transfection
are typically different from forward transfection, and optimal
conditions should be empirically determined for your cells.
The optimization experiment should include two to three cell densities
and a range of DharmaFECT transfection reagent volumes. Our
recommendations for the different components in the transfection
optimization experiment are as follows:

2 Appendix•

Optimization of transfection conditions for delivery of
crRNA:tracrRNA•To obtain the highest transfection efficiency of the Edit-R crRNA:tracrRNAcomponents with minimal effects on cell viability, we recommend carefully
optimizing transfection conditions for each cell line using a positive control
crRNA. The transfection optimization can be easily performed in a 96-wellformat allowing for testing of multiple transfection conditions. Transfection

0.01 to 0.4 μL/well of DharmaFECT 1, 2, 3, or 4 transfection reagent
in a 96-well plate.
25 nM positive control (PPIB or DNMT3B) crRNA:tracrRNA per well
(recommended range 20 nM-50 nM).

Use Table 2 for guidance in preparation of samples for 96-well transfection
optimization.

Table 2. Preparing samples for 96-well transfection optimization. Mix the diluted crRNA:tracrRNA and diluted transfection regent and incubate the transfection mixture for 20 minutes. Transfer
the transfection mixture to a well of a tissue culture plate and add 80 μL of cell suspension in growth medium at the desired cell density. This will bring the total volume per well to 100 μL and theconcentration of crRNA:tracrRNA complex to 25 nM. Volumes (μL) are shown per ONE well of a 96-well plate; for triplicate wells multiply all values by 3.5 to have sufficient volume for three wells
and to account for pipetting error. For the diluted transfection reagent prepare a larger volume to enable accurate pipetting of the small volumes that are required per one well.

Tube 2: Diluted DharmaFECT
transfection reagent

Tube 1: Diluted crRNA:tracrRNATransfection condition

Final reagent volumes

DharmaFECT
transfection reagent
amount per well

Serum-free
medium (µL)

Volume of
1 µM crRNA(µL)

Volume of
1 µM tracrRNA(µL)

Serum-free
medium (µL)

Volume ofDharmaFECT
transfection
reagent (µL)

Transfection
mixturevolume (µL)

Cellsuspension in
Growthmedium (µL)

Total volume
per 96-well(µL)

0.01 µL/well

5

2.5

2.5

9.99

0.01

20

80

100

0.025 µL/well

5

2.5

2.5

9.975

0.025

20

80

100

0.05 µL/well

5

2.5

2.5

9.95

0.05

20

80

100

0.1 µL/well

5

2.5

2.5

9.9

0.1

20

80

100

0.2 µL/well

5

2.5

2.5

9.8

0.2

20

80

100

0.3 µL/well

5

2.5

2.5

9.7

0.3

20

80

100

0.4 µL/well

5

2.5

2.5

9.6

0.4

20

80

100

Untreated (0 µL/well)

10

0

0

10

0

20

80

100

At 48-72 hours post-transfection, perform a cell viability assay todetermine the highest lipid concentration that has minimal cell toxicity(≥ 80% cell viability is preferred). After assaying for cell viability, we
recommend that you carefully wash the cells once with PBS and proceedwith a gene editing assay (see below) to determine the condition that
produces the best editing efficiency. Use these optimal conditions for
subsequent transfection of your selected Cas9-expressing cell lines with
the Edit-R crRNA:tracrRNA.

3 Frequently asked questions

Gene editing assay recommendations
A commonly used method for detection of insertions and deletions(indels) in a cell population is a DNA mismatch detection assay suchas using T7 Endonuclease I (T7EI). The mismatch detection assay canbe performed on either purified genomic DNA or whole cell lysate.
For a detailed protocol see https://dharmacon.horizondiscovery.com/
uploadedFiles/Resources/crrna-positive-controls-protocol.pdf.

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How should I store my crRNA and tracrRNAs?
RNA oligonucleotides should be stored at -20 °C or -80 °C in a non-frost
free freezer, either as a dried pellet or resuspended in an RNase-free
10 mM Tris solution buffered to pH 7.4 to help with stability duringfreeze-thaw cycles. We recommend that RNA oligonucleotides beresuspended to a convenient stock concentration (Table 3) and stored insmall aliquots to avoid multiple freeze-thaw cycles. RNA oligonucleotides
should not go through more than four to five freeze thaw cycles. If
degradation is a concern, the integrity of the RNA oligonucleotides canbe evaluated on an analytical PAGE gel.

3

Table 3. Making stock solutions of crRNA and tracrRNA.

crRNA or tracrRNAamount (nmol)

Volume (μL) of 10 mM Tris pH 7.4 to be added fordesired final concentration
100 μM stock

10 μM stock

0.5

-

50

2

20

200

5

50

500

20

200

2000

50

500

Exceeds tube volume**When tube volume is exceeded, make a 100 μM stock and dilute it 1:10 to obtain the
10 μM stock.

What is the stability of the Edit-R crRNA and tracrRNAs?
Dried RNA oligonucleotide pellets are stable at room temperature for
two to four weeks, but should be placed at -20 °C or -80 °C for long-term
storage. Under these conditions, the dried crRNA and tracrRNAs will
be stable for at least one year. Once resuspended the RNAs are stable
for at least one year when stored at -20 °C or -80 °C in a non-frost free
freezer. Maintaining sterile, RNase- and DNase-free conditions is always
recommended as a critical precaution.

Can I use my siRNA transfection protocols to transfect Edit-R
synthetic crRNA and tracrRNAs?
Previously optimized protocols to transfect synthetic siRNA into yourcells of interest can be a good starting point for transfection of synthetic
crRNA:tracrRNA.

Can I use a different transfection reagent other than
DharmaFECT transfection reagents to deliver the Edit-R
components into my cells?
We cannot predict the performance of other transfection reagents,
nor can we troubleshoot experiments performed with any reagent
other than DharmaFECT transfection reagents. However, other suitabletransfection reagents designed for RNA transfection could be utilized
provided transfection conditions are carefully optimized for each cell lineof interest.

Can I co-transfect arrayed synthetic crRNA:tracrRNA with
the Edit-R Cas9 Nuclease Expression plasmids?
You can perform genome engineering by transient transfection of thesynthetic crRNA:tracrRNA with the Edit-R Cas9 Nuclease Expression
plasmids using DharmaFECT Duo transfection reagent . However,for performing phenotypic analysis in the cell population in a highthroughput manner, we have found that crRNA:tracrRNA transfection ina cell line that stably expresses Cas9 nuclease produces higher efficiency
gene editing in the cell population with lower toxicity associated with thetransfection.

If you have any questions, contact

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specified. ©2018 Horizon Discovery Group Company—All rights reserved. First published January 2016. UK Registered Head Office: Building 8100,
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