TECHNICAL MANUAL

Dharmacon™ Edit-R™
CRISPR-Cas9 geneengineering withlentiviral Cas9 particles
and synthetic guide RNAs

Contents
1. Introduction 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 . Gene editing using Edit-R Lentiviral Cas9 Nuclease particles and synthetic guide RNAs......... 4
Edit-R Lentiviral Cas9 Nuclease Expression vectors.................................................................................................. 5
Edit-R synthetic guide RNA................................................................................................................................................. 6
3. Gene editing using Edit-R Lentiviral Cas9 Nuclease particles and synthetic guide RNAs......... 6
Edit-R CRISPR-Cas9 gene engineering materials required..................................................................................... 6
Additional materials required............................................................................................................................................ 7
Generation of stable cell line expressing Cas9 nuclease......................................................................................... 7
Blasticidin selection......................................................................................................................................................... 7
FACS selection.................................................................................................................................................................... 8
Transduction of cells with Edit-R Lentiviral Cas9 Nuclease Expression particles...................................... 8
Transfection of synthetic guide RNA............................................................................................................................10
4. Packaging Edit-R Lentiviral Cas9 Nuclease plasmids into particles........................................... 11
5. Appendix.......................................................................................................................................... 11
Optimization of transfection conditions for delivery of Edit-R synthetic guide RNA................................. 11
Gene editing assay recommendations........................................................................................................................12
Volume of medium per surface area in culture dishes..........................................................................................12
Stability and storage...........................................................................................................................................................12
Lentiviral particles...........................................................................................................................................................12
Plasmid DNA.....................................................................................................................................................................12
Synthetic guide RNA......................................................................................................................................................12
6 Frequently asked questions........................................................................................................... 13
7. References........................................................................................................................................ 14
8. Lentiviral particle product safety level information.................................................................... 15
9. Limited use licenses......................................................................................................................... 16

Edit-R CRISPR-Cas9 gene engineering withlentiviral Cas9 and synthetic guide 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 protein) 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, thepre-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 (theCRISPR-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.1

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 CRISPRCas9 system have been well studied and adapted for genome engineering in mammalian cells. In S. pyogenes, only threecomponents are required for targeted DNA cleavage at specific target sites adjacent to a PAM:2 (1) The endonuclease
Cas9, programmed by (2) a mature crRNA processed from transcription of the CRISPR locus/array which complexes
with (3) another CRISPR locus-encoded RNA, the trans-activating CRISPR RNA (tracrRNA, Figure 1A).3 Alternatively, thecrRNA can be fused to the tracrRNA creating a chimeric structure termed a single guide RNA (sgRNA, Figure 1B).2
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.4,5 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 ofgenomic DNA 5' of the PAM (red).

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2

Gene editing using Edit-R Lentiviral Cas9 Nuclease particles
with synthetic guide RNAsThe Edit-R platform includes these critical components based on the natural S. pyogenes system: a vector for
Cas9 expression and chemically-synthesized guide RNA designed to the target site of interest. Synthetic guide RNA
can be either a synthetic single guide RNA or a tracrRNA complexed with a gene-specific synthetic crRNA. To facilitate
rapid generation of cell lines that constitutively express Cas9 nuclease, the Edit-R lentiviral Cas9 expression vector ispackaged into particles, purified and concentrated for direct transduction. Subsequent transfection of synthetic guideRNA into Cas9-expressing cell lines results in a higher percentage of edited cells in comparison to co-transfection of a
Cas9 expression plasmid DNA with guide RNA. Figure 2 summarizes a general experimental workflow to generate stable
cell lines expressing the Cas9 nuclease that can then be transfected with synthetic guide RNAs for phenotypic analysis
of gene knockout.

Edit-R
Cas9

Lentiviraltransduction

1
day

1
day

Target cells
Selection withblasticidin or FACS

6-15
days

3-6
weeks

Clonal cell isolationand expansion

Expansion of
selected cell
population

Cas9 clonal cell line

Cas9 cell line
mixed populationorSynthetic
guide RNAtransfection

Synthetic
guide RNAtransfection

3
days

3
days

M

Note

or

C

1

2

3

Gene editing and phenotypic analyses

Figure 2. Gene knockout workflow using the Edit-R Lentiviral Cas9 Nuclease Expression particles with synthetic guide RNA. The workflow describes generating
either a mixed cell population or clonally isolated Cas9-expressing cell line for transfection with guide RNA.

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Edit-R Lentiviral Cas9 Nuclease Expression vectors
The Edit-R Lentiviral Cas9 Nuclease Expression vectors contain a human codon-optimized version of the S. pyogenes
cas9 (csn1) gene and the blasticidin resistance marker (BlastR). The Edit-R Lentiviral Fluorescent Cas9 Nuclease Expression
vectors contain a human codon-optimized version of the S. pyogenes cas9 (csn1) and a fluorescent reporter (mKate2 orTurboGFP™). In both reporter vectors, expression is bicistronic with a 2A peptide sequence linker and under the control ofa single promoter (Figure 3). Reporter genes are placed upstream of the Cas9 coding region rather than the 3' end so thatno extra amino acids are added to the C-terminus, thus preserving the full Cas9 endonuclease activity. A brief descriptionof the lentiviral vector elements are listed in Table 1.
Multiple promoter options are available (Figure 3) for selection of a lentiviral vector with the most active promoter
for specific cells of interest. All Edit-R Lentiviral Cas9 Nuclease Expression vectors are supplied as lentiviral particles(≥ 1 × 107 TU/mL) or dried down, endotoxin-free plasmid DNA, ready for lentiviral packaging.

BlastR T2A

Cas9

WPRE

Edit-R Lentiviral Cas9 Nuclease
AmpR

SV40oripUC ori

SV40 pA

SMARTchoicepromoters

hCMV
mCMV
hEF1α

RRE

mKate2

or

T2A

Cas9

WPRE

TurboGFP™

Edit-R Lentiviral FluorescentCas9 Nuclease
AmpR

SV40 ori pUC ori

3’ SIN LTR

3' SIN LTR

5' LTRRRE

5’ LTR

SMARTchoicepromoters

hCMV
mCMV
hEF1
mEF1
PGK
CAG

SV40 pA

Figure 3. Schematic diagram of the Edit-R Lentiviral Cas9 Nuclease Expression vectors.

U6

mCMV

sgRNA

PuroR

Table 1. Elements of the Edit-R Lentiviral Cas9 Nuclease Expression vectors.

Vector
5' element
LTR

Utility
RRE

Cas9

Human codon-optimized S. pyogenes Cas9 nuclease for cleavage of targeted DNA when programmed with guide RNA

T2A

WPRE

3' SIN LTR

Self cleaving peptide allows for simultaneous expression of two proteins from a single transcript

Blast

Edit-R
Lentiviral sgRNA
Blasticidin resistance marker permits antibiotic selection of transduced mammalian cells

mKate2

Red fluorescent protein reporter enables selection of transduced mammalian cells by FACS

TurboGFP

Green fluorescent protein reporter enables selection of transduced mammalian cells by FACS

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

Human cytomegalovirus, chicken β-actin hybrid promoter

5' LTR

5' Long Terminal Repeat necessary for lentiviral particle production and integration of the construct into the host cell genomeΨPsi packaging sequence allows lentiviral genome packaging using lentiviral packaging systems

RRE

Rev Response Element enhances titer by increasing packaging efficiency of full-length lentiviral genomes

WPRE

Woodchuck Hepatitis Post-transcriptional Regulatory Element enhances transgene expression in target cells

3' SIN LTR

3' Self-inactivating Long Terminal Repeat for generation of replication-incompetent lentiviral particles

SV40 pA

Simian virus 40 polyadenylation signal

pUC oripUC origin of replication

SV40 ori

Simian virus 40 origin of replication

Amp

Ampicillin resistance gene for vector propagation in E. coli cultures

R

R

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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 a published S. pyogenes
tracrRNA sequence.2 It is modified for nuclease resistance and can be used with modified or unmodified Edit-R crRNA.
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 must be immediately upstream of a PAM in the genomic DNA. The
predominant S. pyogenes PAM nucleotide sequence is NGG. Pre-designed crRNAs for gene knockout are available
for human, mouse and rat coding genes on dharmacon.horizondiscovery.com. Custom crRNAs can be designed
and ordered using the Dharmacon CRISPR Design Tool. It is modified for nuclease resistance and can be used withmodified or unmodified Edit-R tracrRNA.
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
sequence3. It is modified for nuclease resistance on both 5' and 3' ends of the molecule. Edit-R synthetic sgRNAs canbe ordered through the Dharmacon CRISPR Design Tool.

3

Protocol for gene engineering using Edit-R Lentiviral Cas9
Nuclease and synthetic guide RNAs
In this workflow, lentiviral Cas9 nuclease is utilized to generate cell lines stably expressing Cas9 nuclease. These cells canthen be transfected with synthetic guide RNAs to achieve efficient gene editing for phenotypic analyses in a populationof cells or in isolated clonal cell lines.

Edit-R CRISPR-Cas9 gene engineering materials required•

Edit-R™ Lentiviral Cas9 Nuclease (choose one):»»

Dharmacon Edit-R Lentiviral Cas9 Nuclease Expression vectors are provided as concentrated, purified lentiviralparticles for immediate transduction or as endotoxin-free plasmid DNA for direct transfection into a packaging
cell line and production of your own lentiviral particles (Table 2).

Table 2. Edit-R Lentiviral Cas9 Nuclease particles and plasmids with SMARTchoice promoter options and selection options

Edit-R™ Lentiviral Cas9 Nuclease Expression Particles & Plasmids with SMARTchoice promoter options
Promoter
hCMV

mCMV

Selection

Particles Cat. No.

Plasmids Cat. No.

BlastRVCAS10124

CAS10136

mKate2

VCAS11869

CAS11877

TurboGFP™

VCAS11868

CAS11876

BlastRVCAS10125

CAS10137
CAS11871

mKate2

VCAS11863

TurboGFP™

VCAS11862

CAS11870

BlastRVCAS10126

CAS10138
CAS11873

mKate2

VCAS11865

TurboGFP™

VCAS11864

CAS11872

mEF1α

BlastRVCAS10127

CAS10139

PGK

BlastRVCAS10128

CAS10140

CAG

BlastRVCAS10129

CAS10141

hEF1α

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Synthetic targeting guide RNA (choose one):»»

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 theDharmacon CRISPR Design Toolb.

tracrRNA, 5, 20 or 50 nmol (Cat #U-002005-XX)

or
»»
•

Edit-R synthetic sgRNA, custom ordering using the Dharmacon CRISPR Design Tool

DharmaFECT Transfection Reagent options. Each formulation is chemically distinct to optimize delivery and viability
across a wide variety of cell types.
»» DharmaFECT 1 (Cat #T-2001-XX)»» DharmaFECT 2 (Cat #T-2002-XX)»» DharmaFECT 3 (Cat #T-2003-XX)»» DharmaFECT 4 (Cat #T-2004-XX)

Additional materials required
The following additional materials are required but not supplied:•

10 mM Tris pH 7.4, nuclease-free buffer (Tris buffer) solution (Dharmacon, Cat #B-006000-100)•

Multi-well tissue culture plates or tissue culture dishes•

Blasticidin S (Fisher Scientific, Cat #BP2647-25; InvivoGen, Cat #ant-bl-1) or flow cytometer•

Assay for assessing cell viability such as Resazurin Cell Viability Reagent•

Assay(s) for detecting gene engineering events in a cell population•

Positive control guide RNA (researcher defined)•

Growth Medium: antibiotic-free cell culture medium (with serum and/or supplements) recommended formaintenance of the cells of interest•

Transduction Medium: the base cell culture medium containing lentiviral particles (with transduction additives
and serum, if necessary)•

Selection Medium: Growth Medium supplemented with the appropriate concentration of blasticidin

Generation of stable cell line expressing Cas9 nuclease
The protocol described here is designed for rapid generation of a cell population where most of the cells have a single
copy of a lentiviral Cas9 nuclease proviral sequence in the genome.
Select the lentiviral Cas9 nuclease vector with the most active promoter in your cell line based on empirical testingor known promoter activity.
Blasticidin selection
The Edit-R Lentiviral Cas9 Nuclease Expression vectors confer resistance to blasticidin in transduced cells. Before
transducing cells, determine the minimum concentration of blasticidin required to kill non-transduced cells between
three and 10 days by generating a blasticidin kill curve. The blasticidin concentration range for many mammaliancells is 2-15 μg/mL.

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FACS selection
The Edit-R Lentiviral Fluorescent Cas9 Nuclease vectors allow creation of stable cell lines through selecting for positive
fluorescent cells using a flow cytometer. For optimal Cas9 gene editing, cell populations with high fluorescence should
be collected. Additionally, single cells with high fluorescence can be sorted into 96-well plates for clonal cell line
generation.
Transduction of cells with Edit-R Lentiviral Cas9 Nuclease Expression particles
The protocol below provides the basic steps for transduction of the lentiviral particles into U2OS cells (as an example)
using serum-free medium in a 24-well plate. Optimal transduction conditions vary widely amongst cell types and must
be determined empirically for each cell line of interest.
		 If a different sized culture dish is used, adjust the number of cells, volumes, and reagent quantities in proportion to thechange in surface area (see Appendix for suggested volumes of Transduction Medium per surface area of culture dishes).

Day 1:
1.

Plate 5 × 104 cells per well in a 24-well plate using Growth Medium.

		 Optimal cell number for plating will vary with growth characteristics of specific cells and should be determined
experimentally. Typically cells should be at 60-80% confluency on the day of transduction.

2.

Incubate cells at 37 °C in a humidified CO2 incubator overnight.

Day 2:
1.

Prepare the Transduction Medium and equilibrate the medium to 37 °C.

2.

Calculate the volume of lentiviral particles needed for transduction at MOI = 0.3 (see Note below).

The functional titer of Edit-R Lentiviral Cas9 Nuclease Expression particles (in HEK293T cells, as determined by qPCR) is
reported on the Certificate of Analysis (C of A). We recommend an MOI of 0.3 (adjusted for relative transduction efficiency
in your cell type) to ensure single integration of the lentiviral Cas9 nuclease. The relative transduction efficiency of yourcell type will likely be lower than that of HEK293T cells.

The equation to calculate a volume of lentiviral stock for a given MOI is:
V = MOI × CN ÷ VT × 1000
Where:
V = volume of lentiviral stock in µL
MOI = desired multiplicity of infection
CN = number of cells in the well at transduction
VT = lentiviral titer in TU/mL (indicated in the Certificate of Analysis) and multipliedby 1000 to convert the volume from mL to µL
For example, for a desired MOI of 0.3 and:• Cell density of 100 000 cells per well at time of transduction• Lentiviral titer = 1 × 107 TU/mL
Then,
V = 0.3 TU/cell × 100 000 cells/well ÷ 1 × 107 TU/mL × 1000 = 3 μL of lentiviral stock per well.

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3.

Thaw the Edit-R Lentiviral Cas9 Nuclease Expression particles on ice.

Lentiviral particles are shipped on dry ice as 25 μL aliquots and must be stored at -80 °C. Under these conditions,lentiviral particles are stable for at least 12 months. Repeated freeze-thaw cycles should be avoided, as this is expected
to negatively affect titer. Once thawed, unused lentiviral particles should be kept on ice, divided into smaller aliquots (ifnecessary) and immediately returned to -80 °C.

4.

Once thawed, gently mix and pipette the calculated volume of lentiviral particles into the base cell medium(no serum) to create the Transduction Medium.

5.

Remove the Growth Medium from the well and add 0.25 mL of the Transduction Medium containing the lentiviralparticles (see Appendix for guidelines on other plate formats).

6.

Incubate cells at 37 °C in a humidified CO2 incubator for 4-6 hours.

7.

Approximately 4-6 hours post-transduction, add an additional 0.75 mL of Growth Medium (with serum) andresume incubation at 37 °C in a humidified CO2 incubator.

If toxicity occurs with your cells, in step 7, replace the medium after 4-6 hours with fresh Growth Medium (with serum).

Days 3-15:
Generation of stably expressing Cas9 cell lines can be achieved by two different methods, depending on the Edit-R Cas9
lentivirus used during the experiment:
Method 1 using Edit-R Lentiviral Fluorescent Cas9 Nuclease1.

At 72 hours post-transduction expand cells into a larger dish and continue to passage until enough cells are collected
for cell sorting (500,000-10 million cells depending on generation of clonal cell line or population).

2.

Prepare cells for FACS analysis according to commonly used protocols.

3.

Sort cells as desired.

We recommend sorting the cells into fractions with low, medium, and high fluorescence expression levels. Additionally,
ensure that each fraction has enough of the total cell population to be easily expanded for downstream applications andtesting. Alternatively, single cells with high fluorescence can be sorted into 96-well plates for clonal cell line generation.

4.

Once the sorted cells are growing normally, expand accordingly to freeze enough aliquots for your experimental
project. Unless single cells were isolated, these cell lines will be a mixed population that on average have a single
integration of Cas9 in their genomes.

Method 2 using Edit-R Lentiviral Cas9 Nuclease with blasticidin selection
1.

At 24-48 hours post-transduction, replace the Transduction Medium with Selection Medium (Growth Medium
containing the appropriate amount of blasticidin).

The appropriate antibiotic concentration is specific to each cell line and should be determined experimentally prior to
selection using a kill curve. Blasticidin usually kills cells between 3 and 10 days, slow growing cells may take longer. If thecells become confluent, split the cells into a larger dish to allow proper blasticidin selection (for example, split cells from
24-well to 6-well culture dishes).

2.

Once the selected cells are growing normally, expand accordingly to freeze enough aliquots for your experimental
project. These cells will be a mixed population that on average have a single integration of Cas9 in their genomes.
9Edit-R CRISPR-Cas9 gene engineering withlentiviral Cas9 and synthetic guide RNAs
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Record the passage number and avoid working with stable cell populations at passage numbers exceeding 10 fromfrozen stock.

Utilize the mixed population of Cas9-expressing cells obtained above for transfection with synthetic guide RNA andsubsequent phenotypic knockout analysis for your gene of interest. If clonal cell lines are required for your application,
we recommend that you isolate clonal cell lines for downstream experiments using protocols appropriate for your cellsof interest.

Transfection of synthetic guide RNA
The following is an example protocol to deliver Edit-R synthetic sgRNA or Edit-R synthetic crRNA complexed withtracrRNA into adherent U2OS cells stably expressing Cas9 in 24-well plates.
Day 1:
1.

Plate 1 × 105 U2OS-Cas9 cells per well in a 24-well plate using Growth Medium.

Optimal cell number for plating will vary with growth characteristics of specific cells and should bedetermined experimentally.

Day 2:
1.

Prepare 2 μM synthetic guide RNA transfection complex from previously prepared 10 μM stocks.
»»

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).
or»»

For synthetic sgRNA add 2 μL of 10 μM synthetic sgRNA to 8 μL of Tris buffer (total volume is 10 μL).

2.

In nuclease-free microcentrifuge tubes prepare the synthetic guide RNA complex by adding 6.25 µL of
2 µM guide RNA complex to 43.75 µL of serum-free medium (see Table 3 for volume recommendations for alternative
plating formats).

3.

Prepare a DharmaFECT 3 working solution in a separate tube by diluting 1 µL of DharmaFECT 3 reagent in
49 µL of serum-free medium and mixing gently. Incubate the tube for 5 minutes at room temperature.

The optimal DharmaFECT Transfection Reagent and concentration varies for different cell lines and is affected by the celldensity. Easy-to-transfect cells and lower cell densities typically require a lower amount of DharmaFECT Transfection
Reagent. For replicates, please prepare sufficient sample volumes for the number of replicates and to account for
pipetting error.

4.

Add 50 μL DharmaFECT working solution to each sample tube containing the crRNA:tracrRNA complex. This
brings the total volume to 100 μL.

5.

Immediately mix by pipetting gently up and down and incubate for 20 minutes at room temperature.

6.

Prepare the Transfection Medium by adding 400 μL antibiotic-free complete Growth Medium to each sample
to bring the total volume in each tube to 500 μL.

7.

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.

8.

Incubate cells at 37 °C in a humidified CO2 incubator for 48 to 72 hours before proceeding with the phenotypic assay
or gene editing analysis (see Appendix). If homogeneous clonal cells are required, proceed with clonal cell isolation
using the preferred protocol.

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Table 3. Suggested volumes per well for transfection of Cas9-expressing cells with 25 nM synthetic guide RNA.

Tube 1: Synthetic guide RNA (µL/well)

4

Tube 2: Diluted DharmaFECT
transfection reagent (µL/well)DharmaFECT (µL)

Serum-free
medium (µL)

Growth medium(µL/well)

Finaltransfection
volume (µL/well)

8.75

0.2

9.8

80

100

6.25

43.75

1

49

400

500

4

12.5

87.5

2

98

800

1000

10

25

175

5

195

1600

2000

Plating format(wells/plate)

Well surface area
(cm2/well)

2 µM synthetic
guide RNA

Serum-free
medium (µL)

96

0.3

1.25

24

2

12
6

Packaging Lentiviral Cas9 Nuclease plasmids into particles
Edit-R Lentiviral Cas9 Nuclease Expression plasmids are Tat dependent and requires a packaging system that expresses
the tat gene. For packaging lentiviral constructs, we recommend the Dharmacon Trans-Lentiviral ORF Packaging System.
For packaging protocols and additional information please consult the product manual.
Edit-R Lentiviral Blast-Cas9 Nuclease Plasmids do not express a fluorescent protein reporter, therefore, after packaging
plasmid DNA, we recommend titering the lentiviral particles produced using a functional lentiviral titration protocol
such as limiting dilution with cell viability assay by crystal violet staining or genomic qPCR assay.14 Edit-R Lentiviral
Fluorescent Cas9 Nuclease Expression plasmids have a fluorescent protein reporter and therefore, can be used todetermine functional lentiviral titer in cells through observing fluorescence.

5

Appendix
Optimization of transfection conditions for delivery of Edit-R synthetic guide RNA
To obtain the highest transfection efficiency of the synthetic guide RNA with minimal effects on cell viability, we
recommend carefully optimizing transfection conditions for each cell line using a synthetic control guide RNA. The
transfection optimization can be easily performed in a 96-well format allowing for testing of multiple transfection
conditions. Transfection conditions that have previously been optimized for small RNA delivery are a reasonable starting
point for guide RNA complex transfection.
The optimization experiment should include two to three cell densities and a range of DharmaFECT Transfection Reagent
volumes. Our recommended ranges for the different components are as follows:•

0.05 to 0.8 μL/well of a 96-well plate DharmaFECT 1, 2, 3 or 4•

25 nM control guide RNA complex per well

At 48 to 72 hours post-transfection, perform a cell viability assay to determine the highest lipid concentration thathas minimal cell toxicity (≥ 70% of cell viability is preferred). After assaying for cell viability, we recommend to carefully
wash the cells once with Phosphate Buffered Saline (PBS) and proceed with gene editing analysis (see below) to
determine the condition that produces good editing efficiency. Use the optimal determined conditions for subsequenttransfection of your selected Cas9-expressing cell lines with the Edit-R synthetic guide RNA.

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Gene editing assay recommendations
The most commonly used method for detection of insertions and deletions (indels) in a cell population is a mismatchdetection assay such as T7 Endonuclease I.10,11,12 When edited cells are expanded and clonal populations isolated, themost commonly used method for confirming gene editing is Sanger sequencing.11

Volume of medium per surface area in culture dishes
Table 4. Suggested volumes of Transduction Medium for different plate formats.

Tissue culture dishSurface area per well (cm2)

Suggested total serum-free
medium volume per well (mL)

100 mm56

5

6 well9.4

1

12 well

3.8

0.5

24 well

1.9

0.25

96 well

0.3

0.05

Stability and storage
Lentiviral particles
Edit-R Lentiviral Cas9 Nuclease Expression particles are shipped on dry ice as 25 μL aliquots and must be stored at-80 °C. Under these conditions, lentiviral particles are stable for at least 12 months. Repeated freeze-thaw cycles should
be avoided, as this is expected to negatively affect titer. Once thawed, unused lentiviral particles should be kept on ice,
divided into smaller aliquots (if necessary) and immediately returned to -80 °C.
Plasmid DNA
Edit-R Lentiviral Cas9 Nuclease Expression plasmid DNA are shipped as dried pellets at room temperature. 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, the reagents are stable for at least one year. Always dissolve plasmid in nuclease-free solution, such as
Tris buffer.
Synthetic guide RNA
Edit-R synthetic guide RNA reagents are shipped as dried pellets at room temperature. Under these conditions, they
are stable for at least four weeks. Upon receipt, synthetic guide RNA should be stored at -20 °C to -80 °C. Under these
conditions, the reagents are stable for at least one year.
Always resuspend synthetic guide RNA in nuclease-free Tris buffer. In solution and stored at -20 °C, the reagent aliquots
are stable for at least one year. Avoid multiple freeze-thaw cycles. We recommend not exceeding four to five freeze-thaw
cycles to ensure RNA integrity.

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6

Frequently asked questions
How do I choose between the various Edit-R Lentiviral Cas9 promoter options?
Choose the promoter option that has been demonstrated, either by your own experimental observations or throughreferences in the published literature, to actively express a transgene in your cells of choice. For optimal experimental
confidence or if such information is not available, consider using multiple lentiviral promoter-Cas9 constructs or selecting
the best promoter empirically using the Dharmacon SMARTchoice Promoter Selection Plate Cat #SP-001000-01.
How should I store my synthetic guide RNAs?
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 (Synthetic guide RNA
resuspension protocol) and stored 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.
Can I use my siRNA transfection protocols to transfect Edit-R synthetic guide RNAs?
Previously optimized protocols to transfect synthetic siRNA into your cells of interest can be a good starting point fortransfection of synthetic guide RNA.
Can I use a different transfection reagent other than DharmaFECT to deliver the Edit-R components into my cells?
We cannot predict the transfection ability of other transfection reagents, nor can we troubleshoot experiments
performed with any reagent other than DharmaFECT Transfection Reagents. However, other suitable transfection
reagents designed for RNA transfection could be utilized provided transfection conditions are carefully optimized
for each cell line of interest.
What is the best way to confirm that my gene is knocked out?
Mismatch detection assays tell you that editing occurred in the cell population. Clonal cell isolation followed by DNA
sequencing of the region of interest and determination of the protein functionality are recommended to confirm thegene knockout.
Can the Edit-R lentiviral Cas9 system be used for gene knockout in non-mammalian organisms, such as flies
or worms?
We have designed the Edit-R Cas9 plasmids and lentiviral particles for mammalian expression (and thus have only tested)in mammalian cells. We cannot predict the efficacy of using Edit-R lentiviral Cas9 particles and Edit-R synthetic guide RNAcomponents, nor can we troubleshoot experiments performed in non-mammalian systems.

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Edit-R CRISPR-Cas9 gene engineering withlentiviral Cas9 and synthetic guide RNAs
Technical Manual

Can I co-transfect the Edit-R synthetic guide RNA components with the Edit-R Lentiviral Cas9 Nuclease Expression
plasmid that I bought as DNA rather than making lentiviral particles?
The Edit-R Lentiviral Cas9 Nuclease Expression vectors are intended to be used as lentiviral particles to generate stable
cell lines expressing Cas9. If you intend to perform genome engineering by transient transfection, we recommend thenon-lentiviral Edit-R Cas9 Nuclease Expression plasmids, which are smaller in size and intended for co-transfection withthe synthetic guide RNAs.
Where can I obtain the Edit-R lentiviral Cas9 expression vector maps?
Cas9 expression plasmid maps can be obtained upon request from Technical Support(ts.dharmacon@horizondiscovery.com) or +1 800 235 9880 toll-free; 303 604 9499).
What filters are recommended for sorting cells using mKate2 or TurboGFP™?
mKate2 has an excitation maximum of 588nm and emission maximum of 633nm. Recommended Omega Optical filter
sets are QMAX-Red, XF102-2, Texas red, or similar. TurboGFP™ has an excitation maximum of 482nm and emission
maximum of 502nm. Recommended filter sets are EGFP, FITC, and other green dyes. Recommended Omega Optical filter
sets are QMAX-Green, XF100-2, XF100-3, XF115-2, and XF116-2.

7

References
1.

D. Bhaya, et al. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense
and regulation. Annu. Rev. Genet. 45, 273-297 (2011).

2.

M. Jinek, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial Immunity.
Science. 337, 816-821 (2012).

3.

E. Deltcheva, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor Nuclease III.
Nature. 471, 602-607 (2011).

4.

P. Mali, et al. RNA-guided human genome engineering via Cas9. Science. 339, 823-826 (2013).

5.

T.R. Sampson and D.S. Weiss. Exploiting CRISPR/Cas systems for biotechnology. Bioessays. 36, 34-38 (2014).

6.

Y. Fu, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.
Nat. Biotechnol. 31, 822-826 (2013).

7.

P.D. Hsu, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827-832 (2013).

8.

T. Wang et al. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 343, 80-84 (2014).

9.

A.R. Bassett et al. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system.
Cell Rep. 4, 220-228 (2013).

10. D.Y. Guschin, et al. A rapid and general assay for monitoring endogenous gene modification.
Methods Mol. Biol. 649, 247-256 (2010).
11. D. Reyon, 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).
12. L. Cong, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339, 819-823 (2013).
13. J.C. Kappes, X. Wu and J.K. Wakefield. Production of trans-lentiviral vector with predictable safety.
Methods Mol. Med. 76, 449-465 (2003).
14. R.H. Kutner, X. Y. Zhang and J. Reiser. Production, concentration and titration of pseudotyped HIV-1-based
lentiviral vectors. Nat. Protoc. 4, 495-505 (2009).
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Edit-R CRISPR-Cas9 gene engineering withlentiviral Cas9 and synthetic guide RNAs
Technical Manual

8

Lentiviral particle product safety level information
This Lentiviral Particle Product Safety Level Information constitutes Product Documentation according to clause 1of the Product Terms and Conditions. It is applicable to the following lentiviral particle products:•

Edit-R™ Lentiviral CRISPR-Cas9 Nuclease Expression particles

The Products are solely for internal research use (as set forth in the Product Terms and Conditions) in laboratories where
the containment measures stated below and in applicable laws and regulations are met. Products may not be used for
diagnostic, therapeutic or other commercial purposes and may not to be administered to humans for any purpose or toanimals for therapeutic purposes. The Products are replication-incompetent, self-inactivating (SIN) and non-pathogenic(do not cause infectious human disease).
Any investigator who purchases Dharmacon lentiviral particle products is responsible for consulting with their
institution’s health and biosafety personnel for specific guidelines on the handling of lentiviral vector particles.
Furthermore, each investigator is fully responsible for obtaining the required permissions for research using and theacceptance of replication incompetent SIN lentiviral vectors and replication-defective lentiviral particles into theirlocal jurisdiction and institution.

For questions concerning the design or production of the products, please contact our Molecular Biology support team.

Tel.:
Fax
Email

+1 800 235 9880 (option 2); 303 604 9499 (option 2)+1 800 292 6088; 303 604 9680
ts.dharmacon@horizondiscovery.com

In the US:
For US guidance on containment for lentiviral vectors, please refer to:
1.

The Recombinant DNA Advisory Committee (RAC) guidelines for research with lentiviral vectors(https://osp.od.nih.gov/wp-content/uploads/Lenti_Containment_Guidance.pdf);

2.

The U.S. Department of Health and Human Services Centers for Disease Control and Prevention andNational Institutes of Health, Biosafety in Microbiological and Biomedical Laboratories (BMBL);

3.

The NIH Guidelines For Research Involving Recombinant DNA Molecules (NIH Guidelines)(http://osp.od.nih.gov/office-biotechnology-activities/biosafety/nih-guidelines).

In the EU:
For the EU directives, please consult the following:
1.

Council Directive 2009/41/EC of the European Parliament and of the Council of 6 May 2009 on the contained
use of genetically modified micro-organisms. (revised version of Directive 90/219/EEC of the European Parliamentand of the Council of 23 April 1990 on the contained use of genetically modified micro-organisms, amended byCouncil Directive 98/81/EC of 26 October 1998); and2.

Council Directive 2001/18/EC of the European Parliament and of the Council of 12 March on the deliberate
release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC

15

In Germany:
Required Containment Measures: The containment requirements as stated in the German Genetic Safety Ordinance(GentechnikSicherheitsverordnung) of Safety Level 2* or higher have been assigned to the handling of the abovementioned lentiviral vector particles.
Please note a higher Security Level might be required if the lentiviral vector particles are used for genetic engineering
operations with other products which require a higher Security Level.
*Safety Level 2: activities of low risk for human health and the environment by the state of scientific knowledge(Stand der Wissenschaft).
For the German regulations, please consult the following:
1.
2.

9

German Genetic Engineering Act (Gentechnikgesetz – GenTG); andGenetic Engineering Safety Ordinance (Gentechnik-Sicherheitsverordnung – GenTSV).

Limited use licenses
The Products, use and applications, are covered by pending and issued patents. Certain Label licenses govern the use of
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

mKate2 and TurboGFP are trademarks of Evrogen JSC. Blasticidin S. is a trademark of Thermo Fisher Scientific, Inc. All trademarks are the propertyof Horizon Discovery Company unless otherwise specified. ©2018 Horizon Discovery Group Company—All rights reserved. First published April
2014. UK Registered Head Office: Building 8100, Cambridge Research Park, Cambridge, CB25 9TL, United Kingdom.

V4-0818

t +44 (0) 1223 976 000 (UK) or +1 800 235 9880 (USA); +1 303 604 9499 (USA)f + 44 (0)1223 655 581
w horizondiscovery.com/contact-us or dharmacon.horizondiscovery.com/service-and-support
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom