PROTOCOL

Dharmacon™ Edit-R™ crRNA Libraries
A protocol for transfection of arrayed crRNA plates for gene knockout ortranscriptional activation

Guidelines for using arrayed crRNA plates forgene knockout or transcriptional activation

Materials• Plates of synthetic crRNAs, up to 2 nmol per well in 96-well plates
or up to 0.5 nmol per well in 384-well plates.

Product description
Libraries of predesigned synthetic crRNAs provide an opportunity toapply the CRISPR-Cas9 system for functional gene knockout analysis,or dCas9-VPR system for transcriptional gene activation, in an arrayed
format. Dharmacon crRNA libraries for functional gene knockout consistof Edit-R synthetic crRNAs which are predesigned using the proprietary
Edit-R CRISPR RNA algorithm. This algorithm was trained on functional
knockout data and has demonstrated the ability to select guide RNAtarget regions more likely to give functional knockout of the protein, notjust create a cut. The algorithm additionally includes specificity scoring
using an internal alignment tool for complete offtarget identification. All
Edit-R CRISPRa crRNAs for transcriptional activation are predesignedwith the algorithm described by Horlbeck et. al1., who performed asystematic study of guide RNAs next to transcriptional start sites (TSS)
to identify features that contribute to CRISPRa activity.
Edit-R crRNA Libraries are available as the following:
Catalog libraries of predefined gene family collections for humanand mouse» Four crRNA per gene as an individual crRNA or a pool of four
crRNA per well, at 0.1 nmol, 0.2 nmol or 0.5 nmol/well.
» Provided in NUNC Polystyrene 96-well V-bottom plates(Cat #249952) or 384-well plates (Thermo Scientific Abgene
Cat #AB-0781) with outer columns left open for inclusionof untreated cells and screen-specific positive andnon-targeting controls.
• Cherry-pick crRNA libraries based on a customer’s gene list.
Learn more or get started.
» Multiple individual crRNAs or a crRNA pool, minimum 20 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) or 384 well plates (Thermo Scientific Abgene
Cat #AB-0781)

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Additional required materials are listed below and are not provided with
crRNA library purchase.
• Edit-R tracrRNA, (Dharmacon, Cat #U-002005-XX). tracrRNA isrequired 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 assessing cell viability such as CellTiter-Blue® Cell
Viability Assay (Promega Corp., Cat #G8081)• Positive control crRNA and detection primers for assessment ofgene editing• Non-targeting control crRNA• Assay-specific positive control crRNA (defined by researcher)• Positive control crRNAs for CRISPRa• Non-targeting control crRNAs for CRISPRa• Growth medium: antibiotic-free cell culture medium (with serum
and/or supplements) recommended for maintenance of the cellsof interest Serum-free and antibiotic-free cell culture medium for
preparation of transfection mix (for example, MEM-RS, HyClone
Cat # SH30564).

For phenotypic analysis with the arrayed synthetic crRNA libraries
for gene knockout we strongly recommend using cell lines that
constitutively express Cas9 nuclease. Transfection of crRNA:tracrRNAinto a cell line that is constitutively expressing Cas9 nuclease results ina higher percentage of edited cells thus allowing for easier downstream
high throughput phenotypic analysis of the edited cell population.
For generation of the Cas9 stable expressing cells please follow
recommendations in the Gene Engineering with Lentiviral Cas9 Particles
and Synthetic CRISPR RNAs manual.
For phenotypic analysis with the arrayed CRISPRa crRNAs libraries forgene activation, we recommend using cell lines that constitutively express
dCas9-VPR transcriptional activator. For generation of the dCas9-VPR
stable cells follow commendations in the Dharmacon Edit-R CRISPRatranscriptional activation system with synthetic guide RNA Technical
Manual. The arrayed CRISPRa crRNA libraries may also be used for geneactivation in cells expressing the dCas9-SAM transcriptional activation
system, in which case Edit-R SAM tracrRNA should be used in place of
Edit-R tracrRNA.
The phenotypic analysis, including assay optimization
and analysis, is cell line and assay-specific and requires
optimization by the researcher.
Guidelines for resuspension of the crRNA and generating
crRNA:tracrRNA plates
1. The crRNA libraries are shipped at ambient temperature as drypellets of RNA in each well and should be stored at –20 °C upon
arrival in a manual defrost or non-cycling freezer. If necessary,
crRNAs can be stored as dry pellets (unopened) at 4 °C for
several weeks.
2. crRNA and tracrRNA are used in equimolar amounts, so be sure topurchase enough tracrRNA for the number and quantity of wells
in your library. Calculate the nmol amount of tracrRNA = #wells × nmol quantity per well. For example for a 0.5 nmol library of
100 wells you will need 50 nmols of tracrRNA (0.5 nmol × 100 wells= 50 nmols of tracrRNA). Make 10 μM working concentration of
tracrRNA in Tris pH7.4 buffer as noted in Table 3 of Appendix.
3. Briefly centrifuge crRNA plates to ensure that the crRNA iscollected at the bottom of the well.
4. Wipe adhesive foil cover with 70% ethanol or other RNasedecontamination solution.
5. Pierce or carefully peel back the foil seal to gain access to wells.
Use caution and avoid shredding the seal.
6. If you are starting with a plate of 0.5 nmol per well, resuspend
arrayed crRNAs to 10 μM solution by adding 50 μL of 10 μM
tracrRNA solution in nuclease-free 10 mM Tris pH 7.4 buffer to
0.5 nmol of crRNA (for different quantities of crRNAs see
Appendix, Table 3). This will generate 10 μM of crRNA:tracrRNAmaster plates.
7. Pipette solution up and down 3–5 times while avoiding introductionof bubbles.
8. Seal the plate and place it on an orbital mixer/shaker for 70–90
minutes at room temperature.
9. Briefly centrifuge plates to collect solution to bottom of the wells.
10. From the master crRNA:tracrRNA plate, generate daughter
crRNA:tracrRNA plates with 2 μM working concentration using
nuclease-free, 10 mM Tris pH 7.4 buffer. This eliminates thesubsequent requirement for pipetting of very small volumes.
11. crRNA:tracrRNA plates may now be used immediately, aliquoted
into single-use plates (5 μL/well is recommended for direct use inlipid transfection—see next section) or stored at –20 °C in a manual
defrost or non-cycling freezer.
12. For storage, seal plates with appropriate adhesive or heat seals.

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Alternatively, the crRNAs can be resuspended in 10 mM Tris
buffer to generate master plates of 10 μM crRNA, and the
tracrRNA can be added during the generation of the daughter
plates. Use equimolar amounts of crRNA and tracrRNA togenerate 2 μM crRNA:tracrRNA working plates.
Transfection protocol of arrayed crRNA:tracrRNA plates
The following is a general protocol for transfection of arrayed
crRNA:tracrRNA libraries using stable Cas9-expressing or dCas9-VPRexpressing mammalian cells in 96-well plates in triplicate at 25 nM final
concentration of the crRNA:tracrRNA complex. Optimal plating density
for each transfection method will depend on growth characteristics ofspecific cell lines and assay requirements and these parameters should
be determined experimentally. Exact parameters for crRNA:tracrRNAtransfection in your cells of interest should be empirically determinedthrough careful optimization prior to experimentation (see Appendix for
Optimization of transfection conditions with crRNA:tracrRNA). Catalog
crRNA library plates are supplied with columns 1 and 12 empty to allow
addition of researcher-defined controls. We suggest including thefollowing controls:
1. Untreated cells
2. Positive control crRNA3. Non-targeting crRNA (negative control)
The protocol is provided for transfection of one arrayed crRNA plate intriplicate for a final 25 nM concentration of the crRNA and tracrRNA.
Calculations are done for quadruplicates providing excess volume forthe ease of pipetting. This protocol is written for a direct use of thedaughter plates containing 5 μL of 2 μM crRNA:tracrRNA complex
for preparation of the transfection mix. This protocol uses the least
amount of pipetting and liquid-handling steps.
Positive and negative crRNA controls can be added toempty wells of the V-bottom transfection mix plate(columns 1 and 12 in catalog libraries).
1. Prepare transfection reagent working solution by diluting thetransfection reagent stock solution in serum-free medium.
Preparing 9 mL volume will allow for transfection 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 0.1 µL per well of cells, add 48 µL of transfection
reagent stock solution to serum-free medium for a total volume of
9 mL See table 1 for additional volumes in plate format, or table 2
for individual well recommendations.
2. Add 75 μL of transfection reagent working solution to each wellof 96-well V-bottom daughter plate containing 5 μL of 2 μM
crRNA:tracrRNA complex. This brings the total volume to 80 μL
and the concentration of the crRNA and tracrRNA to 250 nM.
3. Immediately mix by pipetting gently up and down and incubate for
20 minutes at room temperature.
4. Briefly mix the transfection mix (by pipetting) in the V-bottom
plates after 20 min incubation.
5. Add transfection mix to your cells.
a. For the Forward transfection method, seed cells a day inadvance. Before transfection replace the media with 80 μL of
fresh cell media and add 20 μL of transfection mix from the
96-well V-bottom transfection mix prepared in step 3 plate tocorresponding wells of the 96-well tissue culture plate. This
will bring the volume to 100 μL and the final concentration of
the crRNA:tracrRNA complex to 25 nM. Repeat this step for theother two plates to obtain triplicates.

b. For the Reverse transfection method, add 20 μL of the transfection
mixture to each well of three new 96-well tissue culture plates
and add 80 μL of the cell suspension prepared in step 4b toeach well of the three 96-well tissue culture plates. This willbring the volume to 100 μL and the final concentration of the
crRNA:tracrRNA complex to 25 nM.
6. Incubate transfected cells at 37 °C in a humidified CO2 incubatorfor 48–96 hours before proceeding with the phenotypic assay orgene analysis.
Table 1. Preparation of different concentration working solution of transfection
reagent for transfection of one arrayed crRNA plate in triplicate. The
highlighted row indicates the experimental conditions used in steps 1–3, above.

Transfection reagent volume per well of cells (μL)

Transfection reagent volume (μL)

Serum-free medium
volume (mL)

0.01

6

9

0.025

12

9

0.5

24

9

0.1

48

9

0.2

96

8.9

0.3

144

8.9

0.4

192

8.8

0.5

240

8.8

0.6

288

8.7

line using a positive control crRNA. The transfection optimization canbe easily performed in a 96-well format allowing for testing of multiple
transfection conditions. Transfection conditions that have previously
been optimized for siRNA delivery are a reasonable starting point for
crRNA:tracrRNA transfection optimization. Cell-type specific guidelines
for the four DharmaFECT formulations can be found in the
DharmaFECT Cell Type Guide.
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:• 0.05 to 0.8 μL/well of DharmaFECT 1, 2, 3, or 4 in a 96-well plate• 25 nM positive control (PPIB or DNMT3B) crRNA:tracrRNA per well
(recommended range 20 nM to 50 nM).
Use Table 2 for guidance in preparation of samples for 96-welltransfection optimization.
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 andproceed with either a gene editing assay for the gene knockout
libraries (see below) to determine the condition that produces thebest editing efficiency or RT-qPCR analysis for the CRISPRa libraries
to determine the conditions that produce the best transcriptional
activation (see assay recommendation below).Use these optimal
determined conditions for subsequent transfection of your selected
Cas9 expressing cell lines with the Edit-R crRNA:tracrRNA.

Appendix

Gene editing assay recommendations for the CRISPR
knockout libraries
A commonly used method for detection of insertions and deletions(indels) in a cell population is a mismatch detection assay using
T7 Endonuclease I (T7EI). These assays can be performed oneither purified genomic DNA or whole cell lysate. A detailed
protocol is provided.

Optimization of transfection conditions for delivery of
crRNA:tracrRNATo obtain the highest transfection efficiency of the Edit-R
crRNA:tracrRNA components with minimal effects on cell viability, we
recommend carefully optimizing transfection conditions for each cell

Table 2. Preparing samples for 96-well transfection optimization. Volumes (μL) are shown per ONE well of a 96-well plate; for triplicate wells multiply all valuesby 4 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.

Tube 1: crRNA:
tracrRNA(µL/well)Tube 2: Diluted DharmaFECT
Transfection reagent (µL/well)

Transfection condition
DharmaFECT volume(μL/well)

2 µM crRNA:
tracrRNA (µL)

Serum-free
mediumFinal reagent volumes

DharmaFECT
Transfection reagent

Transfection Mix
volume (µL)

Growth Medium orcell suspension (µL)

Total volume
per 96-well (µL)

0.01

1.25

18.74

0.01

20

80

100

0.025

1.25

18.73

0.025

20

80

100

0.05

1.25

18.70

0.05

20

80

100

0.1

1.25

18.65

0.1

20

80

100

0.2

1.25

18.55

0.2

20

80

100

0.3

1.25

18.45

0.3

20

80

100

0.4

1.25

18.35

0.4

20

80

100

0.5

1.25

18.25

0.5

20

80

100

0.6

1.25

18.15

0.6

20

80

100

Untreated

0

20.00

0

20

80

100

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Gene expression analysis recommendations for CRISPRa libraries
RNA can be isolated using different methods per manufacturer’s
instructions. Quantitative RT-qPCR analysis can be performed using
gene expression assays according to manufacturer’s instructions.
Use the expression of a housekeeping gene for normalization of
the expression of the gene of interest. Follow best practices for
RT-qPCR analysis with appropriate number of technical replicatesand proper controls.

Frequently asked questions
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 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) andstored in small 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 can be evaluated on an analytical PAGE gel.
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 for desired final concentration
100 μM stock

10 μM stock

4 μM stock

0.1

*

_

25

0.25

*

25

62.5

0.5

*

50

*

2

20

200

*

5

50

500

*

20

200

2000

*

50

500

*

*

200

2000

*

*

* not recommended due to either small volume for proper mixing or large volume
that exceeds the tube (well) volume When tube volume is exceeded, make thelarger stock and make the dilution with 10 mM Tris buffer pH7.4 in a separate
tube (plate) 4 μM stock is provided so that mixing of equal volumes of crRNAand tracrRNA can generate the working dilution of 2 μM crRNA:tracrRNA for lipid
mediated transfection.

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 tracrRNAand crRNAs will be stable for at least one year. Maintaining sterile,
RNase- and DNase-free conditions is always recommended as a
critical precaution.

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Can I use my siRNA transfection protocols to transfect
Edit-R synthetic crRNA and tracrRNAs?
Previously optimized protocols to transfect synthetic siRNA into
your cells of interest can be a good starting point for transfectionof 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, othersuitable transfection reagents designed for RNA transfection could be
utilized provided transfection conditions are carefully optimized foreach cell line of interest.
Can I co-transfect arrayed synthetic crRNA:tracrRNA with the
Edit-R Cas9 Nuclease Expression plasmid?
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
in a cell line that stably expresses Cas9 nuclease produces higher
efficiency gene editing in the cell population with lower toxicity
associated with the transfection. If the generation of Cas9 stable cells
is not possible we recommend co-delivery of the crRNA:tracrRNA witheither Cas9 mRNA or Cas9 protein.
Can I co-transfect the Edit-R CRISPRa crRNA:tracrRNA components
with the Edit-R CRISPRa Lentiviral dCas9-VPR expression plasmid?
We do not recommend doing a co-transfection of Edit-R LentiviraldCas9-VPR Expression plasmids with synthetic guide RNAs due to thetransient nature of transcriptional activation with the non-expressed
guide RNAs and the longer time necessary to perform blasticidin
selection (typically > 5 days) to remove the non-transfected cells.
For transient expression experiments, or to avoid the use of lentiviralparticles, we recommend doing a co-transfection of the Edit-R sgRNA
plasmid with the Lentiviral dCas9-VPR expression plasmid which thenallows for selection of both components.
Can I use Edit-R CRISPRa crRNA in the dCas9-SAM system?
Yes. Synthetic Edit-R CRISPRa crRNA may be used with synthetic
Edit-R SAM tracrRNA for activation in cells expressing thedCas9-SAM system.

Can I use the Edit-R SAM tracrRNA with the VPR system?
No. Because of the added MS2 aptamer sequence, Edit-R SAM
tracrRNA is specifically designed for use in activation experimentswith cells expressing the SAM components dCas9-VP64 andMS2-p65-HSF1. If working in the dCas9-VPR system, the standard
Edit-R tracrRNA (Cat# U-002005-XX) should be used for synthetic
crRNA: tracrRNA experiments.
Can I co-transfect arrayed synthetic crRNA:tracrRNA with the
Edit-R dCas9-VPR mRNA?
Yes. dCas9-VPR mRNA can be used with arrayed synthetic
crRNA:tracrRNA for activation in cells not stably expressing thedCas9-VPR system.
What plate layout can I expect for my library?
Catalog 96-well libraries are fulfilled with the following plate layout:
80-wells per plate, columns 1 and 12 left empty. Catalog 384-welllibraries are fulfilled with one of the following plate layouts:a) 280-wells per plate, rows A and P, plus columns 1, 2 and 23,
24 left emptyORb) 320-wells per plate, columns 1, 2 and 23, 24 left empty
NOTE: Please refer to the platemap provided with order for yourprecise layout, and contact Technical Support with any questions.

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