TECHNICAL MANUAL

Dharmacon™ Edit-R™
CRISPR-Cas9 geneengineering with Cas9
nuclease expression
plasmids and synthetic
guide RNAs

Contents
1 I. ntroduction to the CRISPR-Cas9 system for gene engineering................................................... 3
CRISPR-Cas: an adaptive immunity defense mechanism in bacteria and archaea....................................... 3
Engineering a CRISPR-Cas9 platform for mammalian genome editing........................................................... 3
2 Edit-R gene engineering with Cas9 expression plasmids and synthetic guide RNAs................ 4
Edit-R Cas9 Nuclease Expression plasmids................................................................................................................... 4
Edit-R synthetic guide RNA................................................................................................................................................. 5
Edit-R trans-activating CRISPR RNA (tracrRNA)..................................................................................................... 5
Edit-R CRISPR RNA (crRNA)............................................................................................................................................ 5
Edit-R synthetic sgRNA............................................................................................................................................... 5
3. Guidelines for co-transfection of Edit-R gene engineering components.................................... 6
Edit-R materials required for gene engineering using Cas9 expression plasmids
and synthetic guide RNAs................................................................................................................................................... 6
Additional materials required............................................................................................................................................ 7
General protocol for co-transfection of Cas9 expression plasmids and synthetic guide RNAs............... 7
Cell plating........................................................................................................................................................................... 7
Co-transfection.................................................................................................................................................................. 7
Enrichment for gene editing events.......................................................................................................................... 9. Gene editing assay recommendations............................................................................................................................ 9
4. Transfection optimization................................................................................................................ 9
5. Appendix.......................................................................................................................................... 12
Cas9 Nuclease Expression plasmid with blasticidin selection marker.............................................................12
Stability and storage...........................................................................................................................................................13
Edit-R Cas9 Nuclease Expression plasmid.............................................................................................................13
Edit-R synthetic guide RNAs......................................................................................................................................13
DharmaFECT™ Duo Transfection Reagent............................................................................................................13. Frequently asked questions...............................................................................................................................................13
References................................................................................................................................................................................18
6 L. imited use licenses......................................................................................................................... 18

Edit-R Cas9 Expression Plasmids and Synthetic RNAs
Technical Manual

1

Introduction to the CRISPR-Cas9 system for gene engineering
CRISPR-Cas: an adaptive immunity defense mechanism in bacteria and archaea
The CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated proteins) system is an adaptive
bacterial and archaeal defense mechanism that serves to recognize and silence incoming foreign nucleic acids. Upon
infection by bacteriophage or other foreign DNA elements, a host organism can incorporate short sequences from theinvading genetic material, called protospacers, into a specific region of its genome (the CRISPR locus) between shortpalindromic DNA repeats of variable lengths. Multiple spacer-repeat units are clustered at the CRISPR locus to form the
CRISPR array. The entire locus including the CRISPR array is transcribed by RNA polymerase into a primary transcript,
the pre-CRISPR RNA (pre-crRNA), which is then processed into small, mature CRISPR RNAs (crRNAs) such that they
include sequences complementary to the foreign, invading DNA. crRNAs guide a multifunctional protein or protein
complex (the CRISPR-associated or Cas proteins) to cleave complementary target DNA that is adjacent to short sequences
known as protospacer-adjacent motifs (PAMs). Thus, the organism acquires a way to protect itself from subsequent
infection (Bhaya, 2011).

Engineering a CRISPR-Cas9 platform for mammalian genome editing
Many bacterial and archaeal CRISPR-Cas systems have been identified with diverse sets of mechanisms, Cas proteinsand multi-subunit complexes. In particular, the processes and key components of the Streptococcus pyogenes
CRISPR-Cas9 system have been well-studied and adapted for genome engineering in mammalian cells. In S. pyogenes,
only three components are required for targeted DNA cleavage at specific target sites adjacent to a protospacer adjacentmotiff (PAM; Jinek, 2012): (1) The endonuclease Cas9, programmed by (2) a mature crRNA processed from transcriptionof the CRISPR locus/array which complexes with (3) another CRISPR locus-encoded RNA, the trans-activating CRISPR RNA(tracrRNA; Figure 1A; Deltcheva, 2011). Alternatively, the crRNA can be fused to the tracrRNA creating a chimeric structure
termed a single guide RNA (sgRNA; Figure 1B: Jinek, 2012).
Upon site-specific double-stranded DNA cleavage, a mammalian cell can repair such a break through either
non-homologous end joining (NHEJ) or homologous recombination (HR). NHEJ is often imperfect, resulting in small
insertions and deletions (indels) that can cause nonsense mutations resulting in gene disruptions to produce geneknockouts (Mali, 2014; Sampson, 2014). This endogenous DNA break repair process, coupled with the highly tractable
S. pyogenes CRISPR-Cas9 system, allows for a readily engineered platform to permanently disrupt gene function.

A.

B.
PAM

Genomic DNA

Genomic DNA

PAM

Cas9 Nuclease

crRNA

Cas9 Nuclease
3'

5'
3'

5'

tracrRNA

5'
3'

sgRNA

Figure 1. Illustration of CRISPR-Cas9 system. Cas9 nuclease (light blue), programmed by the crRNA (green):tracrRNA (purple) complex (A) or the sgRNA (B), cutting
both strands of genomic DNA 5' of the PAM (red).

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Edit-R Cas9 Expression Plasmids and Synthetic RNAs
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Edit-R gene engineering with Cas9 expression plasmids andsynthetic guide RNAsThe Edit-R reagent workflow using Edit-R Cas9 nuclease expression plasmid and synthetic guide RNAs includes these
critical components required for gene editing in mammalian cells based on the natural S. pyogenes system: a plasmid
for expression of a codon-optimized Cas9 nuclease protein and either (a) a synthetic tracrRNA with a synthetic crRNAdesigned to the gene target of interest or (b) a synthetic single guide RNA designed to the gene target of interest.
All components are co-transfected into the mammalian cell of choice using the DharmaFECT™ Duo Transfection Reagent
to perform gene disruption. Figure 2 summarizes the general experimental workflow. Each component is described
below.
synthetic crRNA:tracrRNASelect guide RNAs
Select three to fivesynthetic guide RNAs
per gene0 daysOrder
Cas9 expression
plasmid, guide RNA,
DharmaFECT Duo
Transfection reagent

Figure 2. General Edit-R gene engineering workflow using
Cas9 expression plasmids and synthetic guide RNAs.

5 daysTransfectorCas9 expression
plasmidsynthetic guide RNAs

2-3
days

3-4
days

Enrichment of edited
cells by FACS

Enrichment of edited cellsby antibiotic selection

Cas9 (mKate2)
Nuclease Expression
Plasmid

Cas9 (PuroR)
Nuclease Expression
Plasmids

Edit-R Cas9 Nuclease Expression plasmids
The Edit-R Cas9 Nuclease Expression plasmids contain a human codon-optimized version of the S. pyogenes Cas9 (Csn1)
gene and either the mKate2 fluorescent reporter (Evrogen, Moscow, Russia) or the puromycin resistance marker (PuroR)under the same promoter (Figure 3A). Placing the mKate2 or puromycin resistance marker at the 5' end of the Cas9
coding region rather than the 3' end ensures that no extra amino acids are added to the C-terminus, thus preserving thefull Cas9 endonuclease activity.
SMARTchoice™ promoter options are available (Figure 3B) so one can choose the plasmid with the most active promoter
for specific cells of interest. All Cas9 nuclease expression plasmids are supplied as dried down, endotoxin-free plasmidDNA, ready for transfection.
A Cas9 expression plasmid utilizing the hCMV promoter and blasticidin resistance marker is also available(see Appendix page 11 for vector map and recommendations on using this plasmid).Noptimization (See page 9).
4mKate2 T2A

Cas9

pA

Cas9
(mKate2)Plasmids
Expression
Edit-R Cas9
Expression
andPlasmid
Synthetic RNAs
AmpR

mKate2 T2A

SMARTchoicepromoters

SMARTchoicepromoters

hCMV
mCMV
hEF1α
mEF1α
PGK
CAG
Cas9

Utility

AmpR

Ampicillin resistancegene for vector propagation in E. coli cultures
R

SMARTchoicepromoters

T2A

pA

pA

AmpR

hCMV
Human
codon-optimized S. pyogenes Cas9 nuclease for cleavage ofmCMV
targeted DNA when programmed with synthetic guide RNAs complex
hEF1α
Monomeric
red fluorescent protein for visual tracking of transfection andmEF1αcell sorting (excitation and emission maxima 588 and 633 nm, respectively)
PGK
Self cleaving
CAG peptide allows for simultaneous expression of mKate2

mKate2

Cas9

Cas9 (PuroR) Expression Plasmid

AmpR

Cas9

hCMV
mCMV
hEF1α
mEF1α
PGK
CAG
PuroR T2A

pA

Cas9 (mKate2) Expression Plasmid

Vectorelement

Technical Manual

Vectorelement

UtilityCas9

Human codon-optimized S. pyogenes Cas9 nuclease for cleavage oftargeted DNA when programmed with synthetic guide RNAs complex

PuroR

Puromycin marker confers antibiotic resistance in mammaliancells for selection of transfected cells

T2A

Self cleaving peptide allows for simultaneous expression of
puromycin and Cas9 protein from a single transcript

AmpR

Ampicillin resistance gene for vector propagation in E. coli cultures

pA

Bovine growth hormone (BGH) polyadenylation signal

and Cas9 protein from a single transcript

Cas9
Puro T2A
pA
Bovine growth hormone (BGH) polyadenylation signal

Figure 3A. VectorCas9
elementsof the REdit-R
Cas9 NucleasePlasmid
Expression Plasmids.
(Puro) Expression

Promoter

Description

AmpR

hCMV

human cytomegalovirus immediate early promoter

mCMV

mouse cytomegalovirus immediate early promoter

hEF1α

human elongation factor 1 alpha promoter

mEF1α

mouse elongation factor 1 alpha promoter

PGK

mouse phosphoglycerate kinase promoter

CAG

chicken beta actin hybrid promoter

Figure 3B. SMARTchoice promoter options
for Cas9 Nuclease Expression Plasmids.

Edit-R synthetic guide RNA
Edit-R trans-activating CRISPR RNA (tracrRNA)
The Edit-R tracrRNA is a chemically synthesized and HPLC-purified long RNA based on the published S. pyogenes
tracrRNA sequence (Jinek, 2012). The Edit-R tracrRNA has been tested for efficient editing in multiple mammaliancell types.
Edit-R CRISPR RNA (crRNA)
The Edit-R crRNA is a synthetic RNA, comprised of 20 nucleotides identical to the genomic DNA target site, or
protospacer, followed by a fixed S. pyogenes repeat sequence that interacts with the tracrRNA. The chosen protospacer
sequence in the target genomic DNA, determined by the end user, must be immediately upstream of a PAM in the
genomic DNA. The predominant S. pyogenes PAM nucleotide sequence is NGG (Figure 1). Pre-designed crRNAs
are available for human, mouse and rat coding genes on dharmacon.horizondiscovery.com and custom crRNA can be
designed and ordered using the Dharmacon CRISPR Design Tool.
Edit-R synthetic single guide RNA (sgRNA)
Edit-R synthetic sgRNA is a 100 nucleotide chimera fusing the crRNA and tracrRNA sequences with a 4 nt tetraloop
sequence (Jinek 2012). It is modified for nuclease resistance on both 5’ and 3’ ends of the molecule. Edit-R synthetic
sgRNAs can be ordered through the Dharmacon CRISPR Design Tool.

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Edit-R Cas9 Expression Plasmids and Synthetic RNAs
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3

Guidelines for co-transfection of Edit-R gene engineering components
Successful co-transfection of Edit-R components (Cas9 Nuclease Expression plasmid and synthetic guide RNA) andsubsequent gene knockout requires careful optimization of delivery conditions with DharmaFECT Duo Transfection
Reagent for each cell line of interest. For general recommendations on optimizing co-transfection conditions, see on
pages 9-11. The protocol below assumes that experimental conditions have been optimized as recommended.

Edit-R materials required for gene engineering using Cas9 expression plasmids andsynthetic guide RNAs
Edit-R CRISPR-Cas9 materials for gene editing can be ordered at dharmacon.horizondiscovery.com.
•

Cas9 Nuclease Expression plasmid with promoter and reporter/marker of choice, 120 µg dried•••

Cas9 Nuclease Expression plasmids with mKate2 fluorescent reporter•

Edit-R hCMV_mKate2-Cas9 Expression Plasmid DNA (Cat #U-004100-120)•

Edit-R mCMV_mKate2-Cas9 Expression Plasmid DNA (Cat #U-004200-120)•

Edit-R hEF1α_mKate2-Cas9 Expression Plasmid DNA (Cat #U-004300-120)•

Edit-R mEF1α_mKate2-Cas9 Expression Plasmid DNA (Cat #U-004400-120)•

Edit-R PGK_mKate2-Cas9 Expression Plasmid DNA (Cat #U-004500-120)•

Edit-R CAG_mKate2-Cas9 Expression Plasmid DNA (Cat #U-004600-120)

Cas9 Nuclease Expression plasmids with PuroR mammalian antibiotic selection marker•

Edit-R hCMV_PuroR-Cas9 Expression Plasmid DNA (Cat #U-005100-120)•

Edit-R mCMV_PuroR-Cas9 Expression Plasmid DNA (Cat #U-005200-120)•

Edit-R hEF1α_PuroR-Cas9 Expression Plasmid DNA (Cat #U-005300-120)•

Edit-R mEF1α_PuroR-Cas9 Expression Plasmid DNA (Cat #U-005400-120)•

Edit-R PGK_PuroR-Cas9 Expression Plasmid DNA (Cat #U-005500-120)•

Edit-R CAG_PuroR-Cas9 Expression Plasmid DNA (Cat #U-005600-120)•

Cas9 Nuclease Expression plasmid with BlastR mammalian antibiotic selection marker, 120 µg dried(Cat #U-001000-120); see Appendix for vector map and recommendations on using this plasmid•(Optional) mKate2 Transfection Optimization Plasmid, 120 µg dried (Cat # U-003000-120);
see Appendix for vector map and recommendations on using this plasmid

Synthetic targeting guide RNA (choose one):
1.

Edit-R synthetic crRNA and tracrRNA oligos:
a. crRNA, predesigned for your gene of interest in a variety of sizes, or designed and ordered using
the Dharmacon CRISPR Design Tool2.
•b. tracrRNA, 5, 20 or 50 nmol (Cat #U-002005-XX)
orEdit-R synthetic sgRNA, custom ordered using the Dharmacon CRISPR Design Tool

Non-targeting Control guide RNA (choose one):
1.

2.

Edit-R crRNA Non-targeting Control (Cat #U-007501-XX, U-007502-XX, U-007503-XX,
U-007504-XX, U-007505-XX)
orCustom synthetic sgRNA non-targeting control ordered using the Dharmacon CRISPR Design Tool
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Edit-R Cas9 Expression Plasmids and Synthetic RNAs
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We recommend testing at least three to five guide RNA designs per gene of interest to identify the one with highest
editing efficiency that also results in complete knockout of functional protein.

•

DharmaFECT Duo Transfection Reagent (Cat #T-2010-01 (0.2 mL); T-2010-02 (0.75 mL); T-2010-03(1.5 mL); or T-2010-04 (1.5 mL × 5 tubes)

Additional materials required
Transfection experiments require standard cell culture reagents and instruments appropriate for maintenance of cells.
The following additional materials are required but not supplied:
•
•
•
•
•
•
•
•

Multi-well tissue culture plates or tissue culture dishes
Antibiotic-free complete medium: Cell culture medium (including serum and/or supplements) recommended formaintenance and passaging of the cells of interest without antibiotic
Assay for assessing cell viability such as Resazurin cell viability reagent
Assay(s) for detecting gene engineering events in a cell population(Optional) Puromycin antibiotic(Optional) Blasticidin S antibiotic(Optional) Flow cytometer, for FACS enrichment
10 mM Tris pH 7.4 nuclease-free buffer (Tris buffer) solution (Cat #B-006000-100)

General protocol for co-transfection of Edit-R Cas9 expression plasmids and synthetic RNAs
The following is a general protocol using DharmaFECT Duo Transfection Reagent to deliver Edit-R Cas9 Nuclease
Expression plasmid, and synthetic guide RNA into cultured mammalian cells. Exact reagent amounts and parameters
for co-transfection should be empirically determined through careful optimization in your cells of interest prior toexperimentation (see Transfection Optimization section on page 9). The protocol below describes delivery conditions in
HEK293T cells in 24-well plate format and is given for illustrative purposes only.
All steps of the protocol should be performed in a laminar flow cell culture hood using sterile technique.
Cell plating
Optimal cell number for plating will vary with growth characteristics of specific cells and should to be
determined empirically.
1.
2.
3.
4.

Trypsinize and count cells.
Dilute cells in antibiotic-free complete medium to the appropriate stock density. For example, HEK293T cells
should be diluted to 100,000 cells in 0.5 mL of medium for plating at 100,000 cells/well in a 24-well plate.
Plate 0.5 mL of cell suspension into each well of a 24-well plate.
Incubate cells at 37 °C with 5% CO2 overnight.

Co-transfection
Table 1. Recommended samples for a gene engineering co-transfection experiment.
Sample namePurposeCas9 Nuclease Expression plasmid only with a Non-targeting control synthetic guide RNA

Negative control: Expression of Cas9 nuclease without targeting RNAs

Cas9 Nuclease Expression plasmid with gene-specific synthetic guide RNA

Gene engineering sample: Expression of Cas9 nuclease programmed byRNAs for targeted double-strand break in gene of interest

Untreated

No treatment control sample: Confirmation of cell viability

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Edit-R Cas9 Expression Plasmids and Synthetic RNAs
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After each reagent addition, mix the contents of each tube by pipetting gently up and down.

5.

Three different samples are recommended for a gene engineering experiment (Table 1).

6.

Prepare a 100 ng/µL Cas9 plasmid working solution by adding 1.2 mL Tris buffer to 120 μg plasmid. Verify the DNA
concentration using UV spectrophotometry at 260 nm, and adjust the volume if necessary to obtain 100 ng/µL.

7.

Prepare 2 μM synthetic guide RNA transfection complex from previously prepared 10 μM stocks (see Appendix)
a. For crRNA:tracrRNA add 2 μL of 10 μM crRNA and 2 μL of 10 μM tracrRNA to 6 μL of Tris buffer (total volume
is 10 μL).
orb. For synthetic sgRNA add 2 μL of 10 μM synthetic sgRNA to 8 μL of Tris buffer (total volume is 10 μL).
In 1.7 mL tubes prepare samples to be transfected as described in Table 2 (columns 2 - 4) for a final 25 nM
concentration of the guide RNA and 1 μg/well of Cas9 expression plasmid.

8.
9.

Prepare a 60 μg/mL DharmaFECT Duo working solution by diluting 120 μL of 1 mg/mL stock DharmaFECT Duo
in 2 mL serum-free medium and mix gently. Incubate tubes for 5 minutes at room temperature.

Table 2. Preparing transfection samples for gene editing experiment in a 24-well plate format.

Sample Name

Serum-free
mediumWorking
guide RNA
solution(2 µM)

Working
Cas9 plasmid
solution(100 ng/µL)

WorkingDharmaFECT
Duo solution(60 μg/mL)

GrowthmediumTotal
volume per
24-well

Cas9 Nuclease Expression plasmid with Non-targeting
control synthetic guide RNA

35

5

10

50

400

500

Cas9 Nuclease Expression plasmid withgene-specific synthetic guide RNA

35

5

10

50

400

500

Untreated

100

0

0

0

400

500

Volumes (µL) for single 24-well samples of HEK293T cells. For replicates, please prepare sufficient sample volumes for the number of replicates and to account for
pipetting error. Exact reagent amounts for co-transfection in other cell lines of interest should be empirically determined through careful optimization prior toexperimentation (see “Transfection Optimization” Section on page 9).

10. Add 50 μL DharmaFECT Duo working solution to each sample tube as shown in Table 2 (column 5). This will
result in 3 μg/well final DharmaFECT Duo concentration. DO NOT add DharmaFECT Duo working solution to theuntransfected control, which should contain serum-free medium only. This brings the total volume to 100 μL
in each tube. Mix by pipetting gently up and down and incubate for 20 minutes at room temperature.
11. Prepare transfection medium by adding 400 μL antibiotic-free complete medium to each sample to bring
the total volume in each tube to 500 μL (columns 6 and 7).
12. Remove medium from the wells of the 24-well plate containing cells and replace with 500 μL of the appropriate
transfection medium to each well.
13. Incubate cells at 37 °C in 5% CO2 for 48 to 72 hours before proceeding with gene editing analysis.
Take an aliquot of the cells for analysis of gene editing (see “Gene Engineering Assay Recommendations” see below).
Enough cells must be retained after gene editing confirmation for clonal cell line generation.
If you cannot detect gene editing or the editing level in your assay is low, you can consider enriching the cell population
as described in the next section.

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Enrichment for gene editing events
Cell populations with increased frequencies of Cas9-induced gene engineering events can be achieved by two different
methods, depending on the Edit-R Cas9 expression plasmid used during the experiment:
a. mKate2-Cas9 expression plasmid for enrichment by FACS analysis
Use cells transfected with mKate2-Cas9 expression plasmid 48 to 72 hours post-transfection. We recommend
performing the transfection experiment in a larger tissue culture dish so that a sufficient number of cells
can be harvested for FACS analysis according to commonly used protocols. We recommend sorting the cells
into fractions with low, medium, and high mKate2 expression levels. Ensure that each fraction has enoughof the total cell population such that desired fractions can be easily expanded for downstream applications
and testing.
mKate2 is a monomeric far-red fluorescent protein with excitation maximum of 588 nm and emission maximumof 633 nm. Recommended Omega Optical filter sets are QMAX-Red and XF102-2 or equivalents. mKate2 can also
be detected using Texas Red filter sets or similar.
b. PuroR-Cas9 expression plasmid for enrichment by puromycin selection
For enrichment by puromycin selection, use cells transfected with PuroR-Cas9 expression plasmid. The
appropriate puromycin concentration is specific to each cell line and should be determined experimentallyprior to selection using a kill curve. We recommend splitting cells 24 to 48 hours post-transfection prior tostarting the puromycin selection. Incubate cells in selection medium for an additional 2 to 4 days.
Since antibiotics are most effective when cells are actively dividing, it is best to split cells such that they are
not more than 25% confluent.

Gene editing assay recommendations
The most commonly used method for detection of indels in a cell population is a mismatch detection assay suchas T7 Endonuclease I
When edited cells are expanded and clonal populations are obtained, the most commonly used method for detecting
gene editing is Sanger sequencing (Reyon, 2012).

4

Transfection optimization
To obtain the highest transfection efficiency of the Edit-R CRISPR-Cas9 components with minimal effects on cell viability,
we recommend carefully optimizing transfection conditions for each cell line. Perform transfection optimization using
the Edit-R mKate2-Cas9 Expression Plasmid with the most active promoter for your cells to find conditions that show
highest fluorescence intensity (excitation and emission maxima 588 and 633 nm, respectively) and > 70% cell viability
compared to untreated cells. The transfection optimization can be easily performed in a 96-well format allowing fortesting of multiple transfection conditions while using low amounts of the plasmid. The identified conditions can be
subsequently scaled up for transfection in a larger tissue culture dish based on the surface area.

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DharmaFECT Duo transfection regent is optimized for delivering both RNA and plasmid into cells. If anothertransfection reagent is used, careful optimization is recommended to confirm both the RNA and plasmid components
are delivered efficiently to the cells.
Table 3. Transfection optimization layout in a 96-well plate format.

Transfection
reagent (μg/well)

Cell density 1
12

3

Cell density 2
4

5

6

7

8

9

10

11

0.2

A

100 ng200 ng

100 ng200 ng0.3

B

100 ng200 ng

100 ng200 ng0.4

C

100 ng200 ng

100 ng200 ng0.5

D

100 ng200 ng

100 ng200 ng0.6

E

100 ng200 ng

100 ng200 ng0.7

F

100 ng200 ng

100 ng200 ng0.8

G

100 ng200 ng

100 ng200 ng

UntransfectedH

12

Plate row and column are indicated in bold letters and numbers. A series of DharmaFECT Duo concentrations are tested in rows A-G. Two different cell densities aretested in the left and right halves of the plate. Two plasmid concentrations are tested, 100 ng/well in columns 1-3 and 7-9 and 200 ng/well in columns 4-6 and 10-12.
Cells in row H are not transfected.

The optimization experiment should include at least two cell densities (in the range of 60 to 80% confluency) anda range of DharmaFECT Duo transfection reagent volumes. Our recommended ranges for the different components
are as follows:•

20 to 80 μg/mL DharmaFECT Duo (corresponding to 0.2 to 0.8 μL/well of a 96-well plate)•

100 to 200 ng of plasmid per well of a 96-well plate

The following is a description of a transfection optimization protocol in a 96-well plate run in triplicate (suggested
experimental layout in Table 3).
1.

Plate cells in a 96-well tissue culture plate one day before the transfection such that the cells are 60 to 80%
confluent on the following day. It is recommended to test at least two cell plating densities.

2.

Prepare Cas9 plasmid working solution at 100 ng/µL by adding 1.2 mL nuclease-free, Tris buffer to 120 μg
plasmid. Verify the DNA concentration using UV spectrophotometry at 260 nm, and adjust the volume ifnecessary to obtain 100 ng/µL.

3.

In separate tubes, prepare seven DharmaFECT Duo working solutions (20 to 80 μg/mL) by diluting 10, 15, 20, 25,
30, 35 and 40 μL of 1 mg/mL stock DharmaFECT Duo transfection reagent into 500 µL serum-free medium andmix gently. The final DharmaFECT Duo amount is 0.2 to 0.8 µg/well of a 96-well plate. Incubate tubes for 5 minutesat room temperature.

4.

To a deep well plate, add serum-free medium and Cas9 plasmid working solution for a total of 10 μL (Table 4).
Then add 10 μL DharmaFECT Duo working solution for a total volume of 20 μL Transfection Mix for each well. Thisis repeated for each DharmaFECT Duo working solution. Mix by pipetting gently up and down and incubate for
20 minutes. DO NOT add plasmid or DharmaFECT Duo working solution to the untransfected control wells, whichshould contain serum-free medium only.

10Edit-R Cas9 Expression Plasmids and Synthetic RNAs
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Table 4. Preparing samples for 96-well transfection optimization.

Sample Name

Serum-free
mediumWorking
Cas9 plasmid
solution(100 ng/μL)

WorkingDharmaFECT
Duo solution(20-80 μg/mL)

GrowthmediumTotal
volume per
96-well

100 ng Edit-R mKate2-Cas9 Nuclease Expression Plasmid

9

1

10

80

100

200 ng Edit-R mKate2-Cas9 Nuclease Expression Plasmid

8

2

10

80

100

Untreated

20

0

0

80

100

Volumes (µL) for single 96-well samples. The amounts are shown per ONE well of a 96-well plate; for triplicate wells multiply all values by 3.5 to have sufficientvolume for three wells and to account for pipetting error.

5.

Prepare transfection medium by adding 80 μL antibiotic-free complete medium to each sample from Step 4
to bring the total volume in each tube to 100 μL.

6.

Remove medium from the wells of the 96-well plate containing cells and replace with 100 μL of the appropriate
transfection medium (created in Step 5) to each well.

7.

Following transfection, assess the cells for mKate2 expression and cell viability to identify the conditions withhighest mKate2 expression and low cell toxicity.
a. At 24 to 48 hours examine the cells by microscopy to determine conditions with highest fluorescence intensity.
b. At 48 to 72 hours post-transfection, perform a cell viability assay to determine the highest lipid concentrationthat has minimal cell toxicity (>70% of cell viability is preferred).

Although cell viability of 70% or higher is ideal, good editing efficiency can sometimes be achieved under transfection
conditions with lower cell viability.
If after performing transfection optimization, the efficiency is low in your cell line of interest, consider performing
enrichment in the subsequent gene editing transfection, as per the protocols provided in the “Enrichment for Gene 		Editing Events” section, see page 9.

8.

Use the optimal determined conditions for your subsequent co-transfection of Cas9 Nuclease Expression Plasmidwith guide RNA. We recommend concentration of guide RNA between 25 nM to 100 nM (typically 25 nM is optimalfor efficient gene editing). The optimized transfection conditions can be scaled up based on the tissue culture
surface area.
mKate2 fluorescence intensity decreases when the Edit-R mKate2-Cas9 expression plasmids are co-transfected withincreasing concentration of crRNA:tracrRNA complex.

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5

Appendix
Cas9 Nuclease Expression plasmid with blasticidin selection marker
The BlastR-Cas9 Nuclease Expression plasmid expresses a human codon-optimized version of the S. pyogenes Cas9
(Csn1) gene from a human cytomegalovirus (hCMV) promoter, and blasticidin resistance (BlastR) under the control of thesimian virus 40 (SV40)Vector
promoter(Figureendotoxin-freeplasmid
DNA isplasmid
provided in ready-to-transfect amounts.
elements
of5).
theTheEdit-R
Cas9 Nuclease
Expression

hCMV
Cas9
pHCSVBlast-Cas9
BlastR

AmpR

SV40

Vector Element

UtilityhCMV

Human cytomegalovirus promoter for strong, constitutive expression of transgenes in mammalian cells

Cas9

Human codon-optimized S. pyogenes Cas9 nuclease cleaves targeted DNA when programmed with synthetic guide RNAs

SV40

Simian virus 40 promoter drives constitutive expression of blasticidin resistance (BlastR) gene

BlastR

Blasticidin resistance marker confers antibiotic resistance in mammalian cells for enrichment of transfected cells

Amp

Ampicillin resistance gene for vector propagation in E. coli cultures

R

hCMV
Vector elements of the Edit-R
Cas9 Nuclease Expression plasmid
mKate2hCMV

pHCSVBlast-mKate
Cas9

Figure 5. Vector elements of the Edit-R Blast-Cas9 Nuclease Expression
Plasmid.
R

BlastRAmp pHCSVBlast-Cas9
SV40

This plasmid can be used in gene engineering
R experiments where
R blasticidin resistance is required for the experiment.
Blast
Amp
SV40
A compatible mKate2 Optimization plasmid is available where the mKate2 fluorescent marker has replaced the Cas9
nuclease (see vector map in Figure 6). This plasmid could be used for optimization of transfection conditions in asimilar manner as described in “Transfection Optimization” Section, see (pages 10-11).

hCMV
mKate2
pHCSVBlast-mKate
AmpR

BlastR

SV40

Vector Element

UtilityhCMV

Human cytomegalovirus promoter for strong, constitutive expression of transgenes in mammalian cells

mKate2

Monomeric red fluorescent protein for visual tracking of transfection (excitation and emission maxima 588 and 633 nm, respectively)

SV40

Simian virus 40 promoter drives constitutive expression of blasticidin resistance (BlastR) gene

BlastR

Blasticidin resistance marker confers antibiotic resistance in mammalian cells for enrichment of transfected cells

AmpR

Ampicillin resistance gene for vector propagation in E. coli cultures

Figure 6. Vector elements of the Edit-R mKate2 Transfection Optimization Plasmid.

12Edit-R Cas9 Expression Plasmids and Synthetic RNAs
Technical Manual

Stability and storage
Edit-R Cas9 Nuclease Expression plasmid
Plasmid DNA reagents are shipped as dried pellets at room temperature (23 °C). Under these conditions, they are stable
for at least four weeks. Upon receipt, plasmid DNA should be stored at -20 °C to -80 °C. Under these conditions, thereagents are stable for at least one year.
Always dissolve plasmid in nuclease-free solution, such as nuclease-free 10 mM Tris pH 7.4.
Edit-R synthetic guide RNAs
Dharmacon Edit-R synthetic guide RNAs reagents are shipped as dried pellets at room temperature (23 °C). Under these
conditions, they are stable for at least four weeks.
Upon receipt, synthetic guide RNAs should be stored at -20 °C to -80 °C. Under these conditions, the reagents are stable
for at least one year.
Always resuspend RNA in nuclease-free solutions, such as 10 mM Tris pH 7.4. In solution and stored at -20 °C, the aliquoted
reagents are stable for at least two years
DharmaFECT Duo transfection reagent
DharmaFECT Duo transfection reagent is shipped on ice packs. If the ice melts during transit there is no risk to the
product. Stability testing demonstrates no loss in product functionality after prolonged storage at warm temperatures(see the DharmaFECT Stability Testing Product Bulletin for additional details).
Upon receipt, store DharmaFECT Duo transfection reagent at 4 °C. DO NOT FREEZE. DharmaFECT transfection reagentsare labeled with a date of manufacture and are good for 24 months when stored as recommended following that date.

Frequently asked questions
What quality control testing is performed for the synthetic tracrRNA and crRNAs?
The Edit-R tracrRNA is purified by HPLC and evaluated by MALDI-TOF mass spectrometry and HPLC to confirm the lengthand purity. Synthesized crRNAs are evaluated with MALDI-TOF mass spectrometry. All synthetic RNAs are quantified byreadings at A260 using a spectrophotometer.
What production method and quality control testing is performed for synthetic sgRNA?
Edit-R synthetic sgRNA is synthesized using Dharmacon 2’-ACE chemistry, purified by size exclusion, deprotected, anddesalted. The synthetic sgRNA is then identity confirmed by MALDI-TOF mass spectrometry and is quantified by readings
at A260 using a spectrophotometer. Finally, the appropriate quantity is dried down and packaged in a 1.5 mL tube or
96-well plate. For different purification options and larger amounts of synthetic sgRNA these can be custom orderedthrough the single stranded RNA synthesis tool.
Do crRNAs synthesized using Dharmacon™ 2'-ACE chemistry need to be HPLC-purified for gene editing in vitro?
The proprietary Dharmacon 2'-ACE RNA synthesis chemistry has very high coupling efficiencies resulting in RNA oligos
of exceptional yield and crude purity. crRNAs are provided desalted and deprotected to the end user. Additional crRNApurification is not necessary for gene editing experiments. Internal testing and successful gene editing experiments were
performed using desalted and deprotected crRNAs.

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I resuspended my RNA in a buffer and there is a slight yellow tint to the solution. Is there something wrong?
No. Deprotection of the bases during oligonucleotide synthesis uses a dithiolate derivative. Sometimes small quantitiesof this material remain in the sample (thus the yellow tint), but it will have no significant negative effect on editing
experiments or cell viability.
What is the stability of the Edit-R synthetic guide RNA?
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 synthetic guide RNA will be stable for at least one year.
Maintaining sterile, RNase- and DNase-free conditions is always recommended as a critical precaution.
How many freeze-thaw cycles can the Edit-R plasmids, and synthetic guide RNAs be subjected to?
We recommend not exceeding four to five freeze-thaw cycles to ensure product integrity.
How should I store my synthetic guide RNA?
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 during freeze-thaw cycles. We
recommend that RNA oligonucleotides be resuspended to a convenient stock concentration (Table 5) and stored insmall aliquots to avoid multiple freeze-thaw cycles. RNA oligonucleotides should not go through more than four tofive freeze thaw cycles. If degradation is a concern, the integrity of the RNA oligonucleotides can be evaluated on
an analytical PAGE gel.
Table 5. Making stock solutions of synthetic guide RNA.

synthetic guide RNAamount (nmol)

Volume (μL) of 10 mM Tris buffer
to be added for desired final concentration
100 µM Stock

10 µM Stock

1

10

100

5

50

500

10

100

1000

20

200

2000

50

500

Exceeds tube volume** When the volume exceeds the tube volume make the 100 μM stock and dilute it 10 times
to obtain the 10 μM stock.

Delivery of my package was delayed and my Edit-R Cas9 Nuclease Expression plasmid, mKate2 Transfection
Optimization plasmid, tracrRNA and/or crRNAs have been at room temperature for a week. Are they OK to use?
Samples are shipped as a dried pellet and are stable for two to four weeks at room temperature. All RNA oligonucleotides
and plasmid DNA should be placed at -20 °C or -80 °C upon receipt.
How are the Edit-R Cas9 Nuclease Expression plasmids, synthetic tracrRNA and synthetic crRNAs shipped?
The Edit-R Cas9 Nuclease Expression plasmid DNA, mKate2 Transfection Optimization plasmid DNA, tracrRNA and crRNAs
are all provided as dried pellets and shipped at room temperature.
What is the formula for spectrophotometric quantification of synthetic crRNA, tracrRNA and sgRNA?
To quantify RNA, use Beer’s Law: Absorbance (260 nm) = (ε)(concentration)(path length in cm), where ε, epsilon, is themolar extinction coefficient (provided on the Product Transfer Form supplied with the crRNA or synthetic sgRNA order;
the extinction coefficient for tracrRNA Cat# U-002005 is 757800). When solved for the unknown, the equation becomes:
Concentration = (Absorbance, 260 nm) / [(ε)(path length in cm)]. When a standard 10 mm cuvette is used, the path
length variable in this equation is 1. If a different size of cuvette is used, e.g., a 2 mm microcuvette, then the path
length variable is 0.2.
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How should I store the Edit-R Cas9 Nuclease Expression plasmids?
Dried plasmid DNA and DNA resuspended in buffer should be stored at -20 °C or -80 °C. Multiple freeze-thaw cycles
should be avoided and strict DNase- and RNase-free conditions should be maintained to prevent degradation of nucleic
acids.
My DharmaFECT Duo transfection reagent arrived and the ice was melted. Is the reagent still ok to use?
All DharmaFECT formulations, including DharmaFECT Duo reagent, have undergone substantial stability and functional
testing, showing excellent performance even following storage at less-than-ideal conditions. The results are detailed in a
product bulletin available for download.
I see a fair amount of cell death after transfection of my cells. What can I do about this?
Extensive cell death following transfection is an indication that delivery conditions need to be further optimized. Basicparameters to consider when optimizing transfection include transfection reagent and cell-specific conditions suchas the amount of transfection reagent, the lot/batch of transfection reagent, duration of transfection, cell passage
number and cell density at transfection. Often decreasing the amount of lipid present during transfection and/or the
total duration of transfection will help minimize the toxic effect to the cells. Additionally, it is not uncommon to observe
some variability from one tube of transfection reagent to another, and this may also represent a source of experimental
variability. If the problem persists, we recommend that other transfection reagents be considered or you may contact
Technical Support (ts.dharmacon@horizondiscovery.com) for additional troubleshooting help.
In what cells can I use the Edit-R Cas9 Nuclease Expression plasmids, and synthetic guide RNAs?
You can use these components in any mammalian cell type that is amenable to transfection and where the promoter
options for Cas9 expression we provide are active.
How do I choose between the various Edit-R Cas9 expression plasmids?
To select one of the Edit-R Cas9 expression plasmids, consider the following:
1) If you prefer to select Cas9-expressing cells using Blasticidin, and the human CMV promoter is active in yourcells of choice, you can choose the Cas9 nuclease expression plasmid with BlastR mammalian antibiotic
selection marker (Dharmacon Cat #U-001000-120).
2) If you have access to a FACS instrument and prefer to sort cells based on expression of a fluorescent marker,
you can choose one of six Cas9-mKate2 nuclease expression plasmids. Because the Cas9-mKate2 nuclease
expression plasmids are offered with a choice of six different promoters, you must choose one based on known
promoter activity in your cells of choice.
3) If you do not have access to a FACS instrument, or you simply prefer to carry out antibiotic selection via
Puromycin treatment, you can choose one of six Cas9-PuroR nuclease expression plasmids. Since these
are also offered with a choice of six different promoters, you should choose the one with an active promoter
in your cells.
How do I select the Cas9 expression plasmid with the correct promoter for my cells?
We provide Cas9 plasmids with multiple promoter options for driving efficient expression of Cas9 nuclease fused toeither the mKate2 reporter gene or puromycin resistance. We strongly recommend using an Edit-R Cas9 expression
plasmid with a promoter which you know is active in your cells, either by your own experimental observations,or through references in the published literature. If such information is not available, testing several Cas9-mKate2
expression plasmid candidates and observing the comparative mKate2 fluorescence intensity can allow you to makean empirical selection.
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When should I use the Edit-R mKate2 Optimization plasmid?
The mKate2 Optimization plasmid is similar to the Edit-R Cas9 nuclease expression plasmid with the Blasticidin resistancemarker, where the mKate2 fluorescent gene sequence has replaced the Cas9 nuclease gene. The mKate2 Optimization
plasmid can be used to perform transfection optimization in a similar manner as described in the “Transfection
Optimization” section of the technical manual. However, if you are working with a Cas9 nuclease expression plasmid, thenwe recommend performing transfection optimization with a corresponding Cas9-mKate2 expression plasmid under thecontrol of a promoter you know to be active in your cells.
Can I use the Edit-R synthetic guide RNAs components with my own preferred Cas9 nuclease expression plasmid?
We have validated the use of Edit-R synthetic guide RNAs and achieved efficient gene editing utilizing the Edit-R Cas9
expression plasmids in mammalian cell lines. Thus we cannot predict the efficacy of, nor can we troubleshoot experiments
performed with, any other Cas9 nuclease expression vector. However, the repeat component of the crRNA sequence andthe entire tracrRNA sequence are derived from the Streptococcus pyogenes CRISPR-Cas9 system, so they very likely can
successfully be used with another S. pyogenes Cas9 nuclease expression plasmid, as long as the expressed Cas9 sequenceis suitably codon-optimized and is under the control of a promoter which is active in your cells of choice. Additionally, youmust be able to efficiently co-transfect your plasmid DNA with the synthetic RNAs.
What are the benefits of using synthetic guide RNAs compared to a single guide RNA (sgRNA) expressed from a vector?
The main benefit of using the synthetic RNA components is that multiple sequences can be ordered and transfected
within a few days. This allows for rapid screening of several target sites within a single gene or knocking out several genes
quickly, as long as your cells are transfectable. Expressed sgRNAs require individual cloning of each target sequence into an
expression plasmid, growing and selecting several clones to QC by sequencing, then prepping and purifying DNA suitable
for transfection. This can be very time-consuming when you wish to test several candidates per gene or would like toknockout multiple genes quickly.
Can I use a different transfection reagent other than DharmaFECT Duo to deliver the Edit-R components into mycells?
DharmaFECT Duo is specifically optimized for co-transfection of plasmid DNA and small RNA. We have validated our
protocols and demonstrated successful gene editing using DharmaFECT Duo to efficiently deliver the Edit-R components.
Thus we cannot predict the co-transfection ability of other transfection reagents, nor can we troubleshoot experiments
performed with any reagent other than DharmaFECT Duo. However, other suitable transfection reagents could be utilized
provided co-transfection conditions are carefully optimized for each cell line of interest.
My DNA mismatch assay shows a low percentage of editing. Why is that?
Mutation analysis assays which utilize mismatch-specific DNA endonucleases such as T7EI (NEB rely on PCR amplification ofa genomic DNA target site and subsequent observation of cleavage commonly by gel electrophoresis. While these assays
are a straightforward approach for detecting insertions and deletions (indels) introduced by
CRISPR-Cas9 gene editing, sensitivity varies between endonucleases and generally underestimatesediting efficiency for several reasons:1.

Cas9 cleavage followed by DNA repair through non-homologous end-joining (NHEJ) results in deletions,insertions, and mutations of various sizes. Mismatch DNA endonuclease cleavage can produce smeared
bands on a gel which are not easily visualized or quantified.

2. Mismatch DNA endonuclease digestion can lead to non-specific cuts that degrade the PCR product andreduce the intensity of the desired bands, especially at longer incubation times.
3. If the CRISPR-Cas9 gene editing generates large inserts or deletions, primer binding sites can be impacted
and the mutation thus will not be detected by the DNA mismatch assay.
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Will selecting edited cells by treatment with Puromycin or Blasticidin increase editing efficiency?
Puromycin- or Blasticidin-selection can enrich for cells in which editing has occurred and increase % editing; however,the degree of improvement greatly depends on optimized delivery conditions. If the transfection efficiency of the Edit-R
components is high, then antibiotic selection will show a modest improvement in editing efficiency. However, if thetransfection efficiency is low, antibiotic selection improves editing efficiency to a greater degree.
Can the Edit-R system be used for gene knockout in non-mammalian organisms, such as bacteria or worms?
We have designed the Edit-R Cas9 plasmids for expression (and thus have only tested) in mammalian cells. Thus we cannot
predict the efficacy of using Edit-R synthetic RNA components, nor can we troubleshoot experiments performed in nonmammalian systems. However, the repeat component of the crRNA sequence and the entire tracrRNA sequence are derived
from the Streptococcus pyogenes CRISPR-Cas9 system, so they very likely can be used with another S. pyogenes Cas9 nuclease
expression plasmid, as long as the expressed Cas9 sequence is suitably codon-optimized andis under the control of a promoter which is active in your cells of choice. Additionally, you must be able to efficiently
co-transfect your plasmid DNA with the synthetic RNAs.
Can I use the Edit-R synthetic RNA components with my own Cas9 nuclease mRNA? Or mutant Cas9 nuclease
plasmids?
We have validated the use of Edit-R synthetic tracrRNA and crRNAs and achieved efficient gene editing utilizing theEdit-R Cas9 expression plasmids in mammalian cell lines. Thus we cannot predict the efficacy of, nor can we troubleshootexperiments performed with, a mutant Cas9 nuclease expression vector or Cas9 mRNA. However, the repeat componentof the crRNA sequence and the entire tracrRNA sequence are derived from the Streptococcus pyogenes CRISPR-Cas9 system,so they very likely can be used with another S. pyogenes-derived Cas9 component that is suitably optimized and sufficiently
generates active Cas9 protein. Additionally, you must be able to efficiently co-transfect your Cas9 mRNA or plasmid DNA
with the synthetic RNAs.
Can the ploidy of my cell line affect the results of my CRISPR-Cas9 gene editing experiment?
When using a CRISPR-Cas9 system to disrupt gene function, it is important to know as much as you can about the gene
you are targeting and your cells of interest. In particular, the ploidy of your cells, the gene copy number and the presenceof any SNPs are important considerations. In normal diploid cells, obtaining mutations on both alleles may be required
for a complete knockout and an observable phenotype. Virtually all cancer lines and many immortalized cell lines exhibit
aneuploidy and as such, mutation of multiple alleles may be necessary. The presence of SNPs and multiple genomic
locations should also be taken into account when designing crRNAs, as they can affect whether a complete knockout canbe achieved. Expansion of clones from single cells followed by sequence validation to verify the desired mutation on allalleles is the most rigorous approach for confirming complete gene knockout.

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References
D. Bhaya, M. Davison, et al. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense
and regulation. Annu. Rev. Genet. 45, 273-297 (2011).
L. Cong, F. A. Ran, et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 339(6121), 819-823 (2013).
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).
Y. Fu, J. D. Sander, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. (2014).
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).
F. Heigwer, G. Kerr, et al. E-CRISP: fast CRISPR target site identification. Nat. Methods. 11(2), 122-123 (2014).
P.D. Hsu, D. A. Scott, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31(9), 827-832 (2013).
M. Jinek, K. Chylinski, et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity.
Science. 337(6096), 816-821 (2012).
P. Mali, L. Yang, et al. RNA-guided human genome engineering via Cas9. Science. 339(6121), 823-826 (2013).
N. K. Pyzocha, F. A. Ran, et al. RNA-Guided Genome Editing of Mammalian Cells. Methods Mol. Biol. 1114, 269-277 (2014)
D. Reyon, C. Khayter, et al. Engineering designer transcription activator-like effector nucleases (TALENs) by REAL
or REAL-Fast assembly. Curr. Protoc. Mol. Biol. 100, 12.15.1‐12.15.14 (2012).
T. R. Sampson, D. S. Weiss. Exploiting CRISPR/Cas systems for biotechnology. Bioessays. 36(1), 34-38 (2014).
T. Wang, J. J. Wei, et al. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 343(6166),
80-84 (2014).

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Limited use licenses
The Products, use and applications, are covered by pending and issued patents. Certain Label licenses govern the useof the products, these can be found at Dharmacon Licensing Statements. It is each Buyer’s responsibility to determine
which intellectual property rights held by third parties may restrict the use of Products for a particular application.
Please review the Label Licenses governing all use of the Products.

If you have any questions, contact

mKate2 is a trademark of Evrogen JSC All trademarks are the property of Horizon Discovery Company unless otherwise specified.. ©2018 Horizon
Discovery Group Company—All rights reserved. First published April 2016. UK Registered Head Office: Building 8100, Cambridge Research Park,
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