TECHNICAL MANUAL

Dharmacon™
Edit-R™ CRISPR Cas9
gene engineering with
Lentiviral Cas9 and sgRNAContents
1

2
3

4
5

6
7
8
9

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
Gene editing using Edit-R Lentiviral Cas9 Nuclease with sgRNA particles ............................................. 4
Edit-R Lentiviral Cas9 Nuclease and sgRNA Expression vectors ................................................... 5
Protocol for gene engineering using Edit-R Lentiviral Cas9 Nuclease and sgRNAs ................................. 6
Edit-R CRISPR-Cas9 gene engineering materials required ......................................................... 6
Additional materials required ...................................................................................... 7
Generation of a stable cell line expressing Cas9 nuclease ......................................................... 7
Blasticidin selection ........................................................................................... 7
FACS ............................................................................................................ 7
Transduction of cells with Edit-R Lentiviral Cas9 Nuclease Expression particles .............................. 9
Transduction of cells with Edit-R Lentiviral sgRNA particles ..................................................... 10
Puromycin selection......................................................................................... 10
Packaging Edit-R Lentiviral Cas9 Nuclease plasmids ......................................................................... 10
Appendix ...................................................................................................................................... 11
Gene editing assay recommendations ........................................................................... 11
Volume of Transduction Medium per surface area in culture dishes ............................................ 11
Stability and storage ............................................................................................. 11
Lentiviral particles ........................................................................................... 11
Plasmid DNA .................................................................................................. 11
Frequently asked questions ............................................................................................................. 12
References .................................................................................................................................... 13
Lentiviral particle product safety level information ........................................................................... 14
Limited use licenses........................................................................................................................ 15

Edit-R Lentiviral Cas9 and sgRNA
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 infection1.

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 PAM2: (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, the crRNA 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 nonhomologous end joining (NHEJ) or homology directed repair (HDR). NHEJ is often imperfect, resulting in small insertions
and deletions (indels) that can cause nonsense mutations resulting in gene disruptions to produce gene knockouts4,5. 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.

Genomic DNA

Genomic DNA

PAM

Cas9 Nuclease

crRNA

5'

Cas9 Nuclease

3'
3'

5'

5'
3'

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

3

Edit-R Lentiviral Cas9 and sgRNA
Technical Manual

2

Gene editing using Edit-R Lentiviral Cas9 Nuclease with
sgRNA particles
The Edit-R Lentiviral Gene Engineering platform includes two critical components based on the natural S. pyogenes
system: a vector for Cas9 expression and a gene-specific vector for sgRNA expression designed to the target site ofinterest. To facilitate rapid generation of cells lines that constitutively express Cas9 nuclease and the sgRNA, the Cas9 andsgRNA vectors are packaged into lentiviral particles, purified and concentrated. Cas9-expressing cells lines can be easily
generated with the Edit-R Lentiviral Cas9 Nuclease Expression particles and subsequent gene knockouts can be obtainedby an additional transduction with sgRNA lentiviral particles or, alternatively, by transfection with synthetic guide RNA
if the cells are amenable to transfection (see Edit-R™ CRISPR-Cas9 Gene Engineering with Lentiviral Cas9 and Synthetic
guide RNA for more information and protocols). Figure 2 summarizes a general experimental workflow to generate
stable cell lines expressing Cas9 nuclease followed by transduction with sgRNA lentiviral particles for phenotypic analysis
of gene knockout.

Mixed cell
population workflow

Clonal cellline workflow

Lentiviraltransduction

day

15
days

Lentiviraltransduction

Cas9

day

Target cells
Selection withblasticidin or FACS

Selection withblasticidin or FACS

Selected cell
population expansion

Clonal cell isolationand expansion

Cas9 cell line
mixed population

6
weeks

Cas9 clonal
line

Lentiviraltransduction

Lentiviraltransduction

day

day
Cas9-expressing
target cells
Selection withpuromycin

15
days

Selection withpuromycin

Clonal cellisolation

Stably expressing Cas9
puromycin-selected
cell population
expansion
Expandclonal cellsGeneknockout
mixed
population

6
weeks

Figure 2. Gene knockout workflow using theEdit-R Lentiviral Cas9 Nuclease with sgRNA system.
Gene editing with Edit-R Lentiviral Cas9 Nucleaseand sgRNA can be done following a mixed cell
populations approach (left side) typically for geneknockout screenings or on isolated clonal cells lines(right side) when a defined genotype is desired or
required on each step for the phenotypic analyses.

Geneknockoutclonal cells

Gene editing and phenotypic analyses
4Edit-R Lentiviral Cas9 and sgRNA
Technical Manual

Edit-R Lentiviral Cas9 Nuclease and sgRNA 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) or fluorescent reporter (mKate2 or TurboGFP). In both
vectors, expression is bicistronic with a 2A peptide sequence linker and under the control of a single promoter (Figure3). Reporter genes BlastR are placed upstream of the Cas9 coding region rather than on the 3' end so that no extra aminoacids are added to the C-terminus, thus preserving the full Cas9 endonuclease activity. A brief description of the lentiviral
vector elements are listed in Table 1. Multiple promoter options are available (Figure 3A and 3B) for selection of a Cas9
nuclease vector with the most active promoter for specific cells of interest.
In the Edit-R Lentiviral sgRNA vector backbone (Figure 3C), the gene-specific sgRNA is expressed under the control
of a human U6 promoter, while expression of the puromycin resistance marker (PuroR) is driven from the mouse CMV
promoter and allows for rapid selection of cells with integrated sgRNA.
All Edit-R Lentiviral Cas9 Nuclease and sgRNA vectors are supplied as concentrated, purified lentiviral particles(≥ 1 × 108 TU/mL).

A.

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

Edit-R Lentiviral Cas9 Nuclease

3’ SIN
LTR

5’
LTR

hCMV
mCMV
hEF1
mEF1
PGK
CAG

B.

C.

Edit-R Lentiviral FluorescentCas9 Nuclease

U6

3’ SIN
LTR

5’
LTR

or

mCMV

Edit-R Lentiviral sgRNA
Figure 3. Schematic map of the Edit-R Lentiviral Cas9 Nuclease
Expression (A) Edit-R Lentiviral Fluorescent Cas9 Nuclease (B) andsgRNA (C) vectors.

Vectorelement

UtilityCas9

Human codon-optimized S. pyogenes Cas9 nuclease for cleavage oftargeted DNA when programmed with a sgRNA

T2A

Self-cleaving peptide allows for simultaneous expression of
blasticidin resistance and Cas9 protein from a single transcript

BlastR

Blasticidin resistance marker permits antibiotic selection oftransduced 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

U6

Human RNA polymerase III promoter U6

PuroR

Puromycin resistance marker permits antibiotic selection oftransduced mammalian cells

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

AmpR

Ampicillin resistance gene for vector propagation in E. coli cultures5

Edit-R Lentiviral Cas9 and sgRNA
Technical Manual

Each Edit-R Lentiviral sgRNA is specific to the gene or genomic site of choice. The crRNA region of the sgRNA is comprisedof 19-20 nucleotides identical to the genomic DNA target site, or protospacer, followed by the non-variable sgRNA
scaffold containing the tracrRNA sequence from S. pyogenes. The chosen genomic DNA target sequence must be
immediately upstream of a PAM. The predominant S. pyogenes PAM nucleotide sequence is NGG. Pre-designed
gene-specific sgRNAs can be ordered by searching for genes of interest on horizondiscovery.com, or customdesigned using the Dharmacon CRISPR Design Tool.

3

Protocol for gene engineering using Edit-R Lentiviral Cas9
Nuclease and sgRNAs
The Edit-R Lentiviral Gene Engineering system utilizes Cas9 nuclease and the sgRNA in a two-step process. First,
Edit-R Lentiviral Cas9 Nuclease Expression particles are utilized to generate cell lines stably expressing Cas9 nuclease.
These cells can subsequently be transduced with Edit-R Lentiviral sgRNA particles to achieve efficient gene editing forphenotypic analyses in a population of cells or in isolated clonal cell lines.

Edit-R CRISPR-Cas9 gene engineering materials required
Edit-R Lentiviral Cas9 Nuclease Expression vectors are provided as concentrated, purified lentiviral particles for immediatetransduction or as endotoxin-free plasmid DNA for direct transfection into a packaging cell line and production of yourown lentiviral particles. Edit-R Lentiviral sgRNA particles are produced upon ordering and provided as concentrated,
purified lentiviral particles for direct transduction.
•

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

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

Promoter

Selection

Particles Cat. No.

BlastRVCAS10124

hCMV

mKate2

VCAS11869

TurboGFP™

VCAS11868

R

Blast

VCAS10125

mKate2

VCAS11863

TurboGFP™

VCAS11862

BlastRVCAS10126

mKate2

VCAS11865

mCMV

hEF1 α•TurboGFP™

VCAS11864

mEF1α

BlastRVCAS10127

PGK

BlastRVCAS10128

CAG

BlastRVCAS10129

Edit-R™ Lentiviral sgRNA particles:»

Custom sgRNA designed using the Dharmacon CRISPR Design Tool or»

Pre-designed Edit-R sgRNA

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Edit-R Lentiviral Cas9 and sgRNA
Technical Manual

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

Multi-well tissue culture plates or tissue culture dishes•

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

Puromycin (Fisher Scientific, Cat #BP2956-100; InvivoGen, Cat #ant-pr-1)•

Resazurin cell viability reagent or similar•

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

Positive control sgRNA lentiviral particles•

Edit-R Lentiviral sgRNA Non-targeting Controls•

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
and/or puromycin.

Generation of a 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 testing or
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 ten days by generating a blasticidin kill curve. The blasticidin concentration range for many mammaliancells is 2-15 μg/mL.
FACS selection
The Edit-R Lentiviral Fluorescent Cas9 Nuclease vectors allow creation of stable cell lines through selecting for positive
fluorescent cells using Fluorescence-Activated Cell Sorting (FACS). For optimal Cas9 gene editing, cell populations with highfluorescence should be collected. Additionally, single cells with high fluorescence can be sorted into 96-well plates for clonalcell 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. Permissivity to lentiviral delivery and 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 the change in surface
area (see Appendix for suggested volumes of Transduction Medium per surface area of culture dishes).

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Edit-R Lentiviral Cas9 and sgRNA
Technical ManualDay 1:
1. Plate 5  104 cells per well in a 24-well plate using Growth Medium
Typically cells should be at 40-80% confluence 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 by equilibrating the base cell culture medium to 37 °C.
2. Calculate the volume of lentiviral particles needed for transduction at MOI = 0.3.
The functional titer of Edit-R Lentiviral Cas9 Nuclease Expression particles (in HEK293T cells, as determined by qPCR) is reported on the
single integration of the lentiviral Cas9 nuclease. The relative transduction efficiency of your cell type will likely be lower than that of
HEK293T cells. Thus, using an MOI of 0.3 based on the HEK293T titer reported on your C of A may be acceptable for generating a stable
cell population with a single Cas9 integration.

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
For example, for a desired MOI of 0.3 and:Lentiviral titer = 1  108 TU/mL
Then,

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 areunused lentiviral particles should be kept on ice, divided into smaller aliquots (if necessary) and immediately returned to -80 °C.

4.

Once thawed, gently mix and pipette the calculated volume of lentiviral particles into the base cell culture 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) and resume
incubation at 37 °C in a humidified CO2 incubator.

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:
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Edit-R Lentiviral Cas9 and sgRNA
Technical Manual

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.

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 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 the cells to freeze a sufficient number of aliquots for yourexperimental project. These cells will be a mixed population that on average have a single integration of Cas9 intheir genomes.

Utilize the mixed population of Cas9-expressing cell line obtained above for transduction with Edit-R Lentiviral sgRNA
particles for phenotypic analysis by knockout of your gene of interest, or isolate clonal cell lines for downstream
experiments using protocols appropriate for your cells of interest if clonal cell lines are desired.

Transduction of cells with Edit-R Lentiviral sgRNA particles
The protocol below is an example of Edit-R Lentiviral sgRNA Expression particles transduction at an MOI of 0.3 into
adherent U2OS cells stably expressing Cas9 nuclease in 24-well plate format. Permissivity to lentiviral delivery and optimaltransduction conditions vary widely amongst cell types and must be determined empirically for each cell lineof interest.
Puromycin selection
The Edit-R Lentiviral sgRNA vector confers resistance to puromycin in transduced cells. Similar to blasticidin selection,
before transducing cells, generate a puromycin kill curve to determine the minimum concentration of puromycin required
to kill non-transduced cells between three and ten days. The puromycin concentration range for many mammalian cells is
1-10 μg/mL.

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Edit-R Lentiviral Cas9 and sgRNA
Technical ManualDay 1:
1.

Plate 5 × 104 Cas9 Nuclease-expressing cells per well in a 24-well plate using Growth Medium.

2.

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

Day 2:
1.

Prepare the Transduction Medium by equilibrating the base cell culture medium to 37 °C.

2.

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

3.

Thaw the Edit-R Lentiviral sgRNA particles on ice.

4.

Once thawed, gently mix and pipette the calculated volume of lentiviral particles into the base cell culture 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) and resume
incubation at 37 °C in a humidified CO2 incubator.

Days 3-7:
1.

At 24-48 hours post-transduction, replace the medium with Selection Medium containing the appropriate amountof puromycin.

Since the Cas9-expressing cells are resistant to blasticidin, addition of blasticidin to the Selection Medium containing puromycin is

24-well to 6-well culture dishes).

2.

Replace the Selection Medium every 2-3 days and monitor the presence of dead cells daily.

3.

Once the cells are growing normally in Selection Medium, expand the cells to freeze a sufficient number of aliquots
for your experimental project or proceed to isolation of clonal cell lines.
a. Expansion of the puromycin/blasticidin-resistant cells at this stage will generate a mixed population of cells having
a single integration of the sgRNA in their genomes, in addition to the single integration of Cas9 nuclease. This
mixed population will carry a variety of insertions and deletions (indels) and can be used for phenotypic analysisof your gene knockout.
b. If a defined mutation genotype is required, proceed to isolation of clonal cell lines using protocols appropriate foryour cells of interest and determine the exact genotype by DNA sequencing.

4

Packaging Edit-R Lentiviral Cas9 Nuclease plasmids
Edit-R Lentiviral Cas9 Nuclease Expression plasmids are Tat-dependent and require a packaging system that expresses thetat 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
fluorescent protein reporter and therefore, can be used to determine functional lentiviral titer in cells through observing fluorescence.

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5

Appendix
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 I11,12,13. When edited cells are expanded and clonal populations isolated, themost commonly used method for confirming gene editing is Sanger sequencing11.

Volume of Transduction Medium per surface area in culture dishes
Table 3. Suggested volumes of Transduction Medium per surface area per well of adherent cells.

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 and sgRNA 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 reagents 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 innuclease-free solution, such as nuclease-free 10 mM Tris pH 7.4.

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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-00100001).
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 necessary to confirm the geneknockout.
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 Nuclease and sgRNA Expression
particles, nor can we troubleshoot experiments performed in non-mammalian systems.
Can I order the Edit-R Lentiviral sgRNA Expression vector as plasmid DNA?
No. Our Edit-R Lentiviral sgRNA Expression vectors are made to each specific customer order and are sold as lentiviralparticles or custom glycerol stocks.
Can I co-transduce the Edit-R Lentiviral Cas9 Nuclease Expression and my sgRNA particles in my cell lines togenerate my gene knockout faster?
Although this is not the recommended protocol, transduction of both the Edit-R Lentiviral Cas9 Nuclease Expression andthe sgRNA particles can be performed at the same time. If co-transduction is necessary, it is recommended to extend thedual antibiotic selection to the longest selection time period previously determined for the individual antibiotics in yourcells. We cannot predict the outcome, nor troubleshoot any issues, resulting from co-transduction of Cas9 and sgRNA
lentiviral particles.
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.

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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, 602607 (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, 220228 (2013).

10. 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).
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. D.Y. Guschin, et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649,
247-256 (2010).
13. L. Cong, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013).

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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 all Dharmacon lentiviral particle products.
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 use and theacceptance of replication-incompetent SIN lentiviral vectors and replication-defective lentiviral particles into theirlocal jurisdiction and institution.
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).
For questions concerning the design or production of the products, please contact our technical support team.
Horizon Discovery Group
2650 Crescent Drive
Lafayette, CO 80026 USA
Tel. +1 800 235 9880; +1 303 604 9499
Fax: +1 800 292 6088; +1 303 604 9680
Email: technical@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 (Recombinant DNA
Advisory Committee (RAC) guidelines);

2.

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

3.

The NIH Guidelines For Research Involving Recombinant DNA Molecules (NIH Guidelines)(https://osp.od.nih.gov/wp-content/uploads/NIH_Guidelines.pdf).
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 ofgenetically modified micro-organisms. (revised version of Directive 90/219/EEC of the European Parliament and of
the Council of 23 April 1990 on the contained use of genetically modified micro-organisms, amended by Council
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.

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Edit-R Lentiviral Cas9 and sgRNA
Technical Manual

In Germany:
Required Containment Measures: The containment requirements as stated in the German Genetic Safety Ordinance(Gentechnik-Sicherheitsverordnung) 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:

91.

German Genetic Engineering Act (Gentechnikgesetz - GenTG); and2.

Genetic 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, contact

mKate2 and TurboGFP are a trademark of Evrogen JSC. All other uses are strictly prohibited. All trademarks are the property of Horizon Discovery
Company unless otherwise specified. ©2018 Horizon Discovery Group Company—All rights reserved. First published July 2015. UK Registered
Head Office: Building 8100, Cambridge Research Park, Cambridge, CB25 9TL, United Kingdom.

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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
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
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