TECHNICAL MANUAL

Dharmacon™ CRISPRmod
CRISPRa transcriptional
activation system withsynthetic guide RNA

Contents
1 Introduction to the CRISPR-Cas9 system for transcriptional activation...................................... 3
2 CRISPRmod CRISPRa workflow........................................................................................................ 4
CRISPRmod CRISPRa Lentiviral dCas9-VPR expression vectors............................................................................ 4
CRISPRmod CRISPRa synthetic guide RNA................................................................................................................... 5
3. CRISPRmod CRISPRa protocol for transcriptional gene activation.............................................. 6
CRISPRmod CRISPRa materials required....................................................................................................................... 6
Additional materials required............................................................................................................................................ 6
Generation of stable cell line expressing dCas9-VPR............................................................................................... 7
Transfection of synthetic CRISPRmod CRISPRa guide RNAs.................................................................................. 9
Packaging CRISPRmod CRISPRa Lentiviral dCas9-VPR plasmids into particles............................................ 11
4. Appendix.......................................................................................................................................... 11
5 Frequently asked questions........................................................................................................... 12
6 . References........................................................................................................................................ 13
7 Lentiviral particle product safety level information.................................................................... 15
8 Limited use licenses......................................................................................................................... 16

CRISPRmod CRISPRa with synthetic guide RNA
Technical Manual

1

Introduction to the CRISPR-Cas9 system for transcriptional activation
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(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, et al., 2011).

CRISPR-Cas9 platform for transcriptional gene activation in mammalian cells
In addition to genome engineering applications in mammalian cells (Jinek et al., 2012), the Streptococcus pyogenes
CRISPR-Cas9 system has been adapted to technologies for transcriptional regulation (Qi et al., 2013, Gilbert et al., 2013,
Cheng et al., 2013). The nuclease activity of the S. pyogenes Cas9 was abolished by point mutations introduced into two
catalytic residues (D10A and H840A) yielding a deactivated Cas9 (dCas9) that maintains the ability to bind to target DNA
when guided by sequence-specific guide RNAs. When the dCas9 is fused to transcriptional regulators and guided to genepromoter regions, it induces RNA-directed transcriptional regulation. CRISPR-Cas9 based technologies for transcriptional
regulation include CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). CRISPRa utilizes dCas9 fused to different
transcriptional activation domains (Cheng et al., 2013, Gilbert et al., 2014, Tanenbaum et al., 2014, Konermann et al., 2015;
Chavez et al., 2015), which can be directed to promoter regions by specifically designed guide RNA (depicted in Fig 1).
The VPR activation system utilizes a fusion of three transcriptional activators (VP64, p65 and Rta) to the C-terminal endof dCas9 and demonstrates a robust gene activation in mammalian systems (Chavez et al., 2015). Unlike other CRISPRa
systems that require expression of the transcriptional activators from separate vectors, the VPR system requires deliveryof just two components to the cells of interest: dCas9-VPR and a guide RNA, making it easier to utilize across different
biological applications.

A.

B.

Rta

Rta

p65

p65

VP64

PAM

Genomic DNA
dCas9-VPR

crRNA

VP64

TSS
Gene

3'

5'
3'

PAM

Genomic DNA

5'

tracrRNAdCas9-VPR

TSS
Gene

5'
3'

sgRNA

Figure 1. Diagram of dCas9-VPR with crRNA:tracrRNA or sgRNA targeting a gene’s promoter region

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A CRISPRa guide RNA can be either synthetic CRISPR RNA (crRNA) complexed with a trans-activating CRISPR RNA(tracrRNA), or a single guide RNA (sgRNA) where the crRNA has been fused to the tracrRNA creating a chimeric structure(Figure 1). This protocol provides guidance for the two-part synthetic approach (crRNA complexed with tracrRNA). For
CRISPRa using expressed single guide RNA (sgRNA), please see this manual.

2

CRISPRmod CRISPRa workflow
To facilitate rapid generation of cell lines that constitutively express dCas9-VPR, the CRISPRmod CRISPRa lentiviraldCas9-VPR expression vector is packaged into particles, purified and concentrated for direct viral transduction.
Subsequent transfection of synthetic or plasmid CRISPRa guide RNA or transduction of lentiviral CRISPRa sgRNA into
dCas9-VPR expressing cells results in target gene activation. Figure 2 summarizes the general experimental workflow.

1
day

1

Lentiviraltransduction

dCas9VPR

Target cells
Selection withblasticidin

6-15
days

Selected cell
population expansion
dCas9-VPR mixed
cell population

3
days

Synthetic crRNA:
tracrRNA transfection

Selection withpuromycin
Figure 2. CRISPR activation
workflow with lentiviral dCas9-VPR
and synthetic crRNA:tracrRNAGene activation and phenotypic analysis

CRISPRmod CRISPRa Lentiviral dCas9-VPR expression vectors
The CRISPRmod CRISPRa Lentiviral dCas9 -VPR expression vectors contain a human codon-optimized version of thecatalytically inactive S. pyogenes cas9 (csn1) gene due to point mutations on the RuvC1 and HNH nuclease domains (D10A
and H840A) and the blasticidin resistance marker (BlastR). Expression is bicistronic with a 2A peptide sequence and under
the control of a single promoter (Figure 3). A brief description of the lentiviral vector elements is provided in Table 1.
Several promoter options are available (Figure 3) enabling the researcher to choose a lentiviral vector with the mostactive promoter for specific cells of interest. All CRISPRmod CRISPRa Lentiviral dCas9-VPR expression vectors are supplied
as lentiviral particles (≥ 1 × 107 TU/mL, ± 20%) or dried down, endotoxin-free plasmid DNA, ready for lentiviral packaging.4

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Table 1. Elements of the CRISPRmod CRISPRa Lentiviral dCas9-VPR expression vectors.

Vector element

UtilitydCas9-VPR

S. pyogenes dCas9-VPR for gene activation of targeted DNA when programmed with a guide RNA

T2A

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

Blast

Blasticidin resistance marker permits antibiotic selection of transduced mammalian cells

hCMV

Human cytomegalovirus immediate early promoter

mCMV

Mouse cytomegalovirus immediate early promoter

hEF1α

Human elongation factor 1 alpha 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

AmpR

Ampicillin resistance gene for vector propagation in E. coli cultures

R

SMARTchoicepromoters

BlastR

T2A

dCas9-VPR WPRECRISPRmod CRISPRa Lentiviral dCas9-VPR
AmpR

SV40 ori

pUC ori

3’ SIN LTR

5’ LTR

RRE

hCMV
mCMV
hEF1α

SV40 pA

Figure 3. Schematic diagram of the CRISPRmod CRISPRa Lentiviral dCas9-VPR expression vectors.

CRISPRmod CRISPRa synthetic guide RNACRISPRmod CRISPRa RNA (crRNA) for transcriptional activation
CRISPRmod CRISPRa crRNA is comprised of a 20-nucleotide synthetic RNA, identical to the genomic DNA target site, or
protospacer, followed by a fixed S. pyogenes repeat sequence that interacts with the tracrRNA. It is modified for nuclease
resistance and must be used with Edit-R tracrRNA. CRISPRmod CRISPRa crRNAs are pre-designed, based on a published
CRISPRa v2 algorithm (Horlbeck et al., 2016) and target genomic regions in the proximity of a transcriptional start site(TSS). The crRNAs are available as four individual crRNAs or a pool of four crRNA for human and mouse protein coding
genes. When more than one TSS exists for a gene, a second set of crRNA reagents is available (labeled P2) to target thealternative start site.
Edit-R trans-activating CRISPR RNA (tracrRNA)
Edit-R tracrRNA is a chemically synthesized and HPLC-purified RNA based on a published S. pyogenes tracrRNA sequence(Jinek, 2012). It is modified for nuclease resistance and can be used with CRISPRmod CRISPRa crRNA for gene activation or
Edit-R crRNA for gene editing.
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3

CRISPRmod CRISPRa protocol for transcriptional gene activation
In this workflow, CRISPRmod CRISPRa lentiviral dCas9-VPR particles are utilized to generate cells stably expressing dCas9VPR in a population of cells or in isolated clonal cell lines. These cells are then transfected with synthetic guide RNAs toobtain transcriptional target gene activation.

CRISPRmod CRISPRa materials required
CRISPRmod CRISPRa Lentiviral dCas9-VPR
CRISPRmod CRISPRa Lentiviral dCas9-VPR expression vectors are provided as concentrated, purified lentiviral particles
for immediate transduction or as endotoxin-free plasmid DNA for direct transfection into a packaging cell line andproduction of your own lentiviral particles. Select the lentiviral dCas9-VPR nuclease vector with the most active promoter
in your cell line based on empirical testing or known promoter activity.
•

CRISPRmod CRISPRa Lentiviral dCas9-VPR particles with your choice of promoter:
CRISPRmod CRISPRa Lentiviral hCMV-Blast-dCas9-VPR particles (Cat # VCAS11918)
CRISPRmod CRISPRa Lentiviral mCMV-Blast-dCas9-VPR particles (Cat # VCAS11920)
CRISPRmod CRISPRa Lentiviral hEF1α-Blast-dCas9-VPR particles (Cat # VCAS11922)•

CRISPRmod CRISPRa Lentiviral dCas9 -VPR Expression plasmids with your choice of promoter:
CRISPRmod CRISPRa Lentiviral hCMV-Blast-dCas9-VPR plasmid (Cat # CAS11914)
CRISPRmod CRISPRa Lentiviral mCMV-Blast-dCas9-VPR plasmid (Cat # CAS11915)
CRISPRmod CRISPRa Lentiviral hEF1α -Blast-dCas9-VPR plasmid (Cat # CAS11916)

CRISPRmod CRISPRa guide RNA•CRISPRmod CRISPRa synthetic crRNA: Predesigned crRNA for activation of your gene of interest
CRISPRmod CRISPRa Human crRNA pool (Cat# P-HUMAN-XX) 5, 10, 20 nmol tubes
CRISPRmod CRISPRa Human Set of 4 crRNA (Cat# PQ-HUMAN-XX) 2, 5, 10, 20 nmol tubes
CRISPRmod CRISPRa Human crRNA (Cat# CA-HUMAN-XX), 2, 5, 10, 20 nmol tubes
CRISPRmod CRISPRa Mouse crRNA pool (Cat# P-MOUSE-XX) 5, 10, 20 nmol tubes
CRISPRmod CRISPRa Mouse Set of 4 crRNA (Cat# PQ-MOUSE-XX) 2, 5, 10, 20 nmol tubes
CRISPRmod CRISPRa Mouse crRNA (Cat# CA-MOUSE-XX), 2, 5, 10, 20 nmol tubes
The above catalog numbers are agnostic; actual products have gene-specific catalog numbers.
Search for your gene.

•

Edit-R tracrRNA, 5, 20 or 50 nmol (Cat #U-002005-05, -20, -50)

DharmaFECT Transfection Reagent for transfection of crRNA:tracrRNAEach DharmaFECT formulation is chemically distinct to optimize delivery and viability across a wide variety of cell types.
DharmaFECT 1 is the most universal reagent that works well across a wide variety of cell lines. Optimal conditions for
siRNA may be used for crRNA:tracrRNA in a given cell line.
DharmaFECT 1 Transfection Reagent (Cat #T-2001-01, 02, 03, 04)
DharmaFECT 2 Transfection Reagent (Cat #T-2002-01, 02, 03, 04)
DharmaFECT 3 Transfection Reagent (Cat #T-2003-01, 02, 03, 04)
DharmaFECT 4 Transfection Reagent (Cat #T-2004-01, 02, 03, 04)
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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)
Positive control CRISPRmod CRISPRa crRNA reagent:
CRISPRmod CRISPRa Human POU5F1 crRNA (Cat #U-009200-01-05 or -20)
CRISPRmod CRISPRa Human POU5F1 crRNA pool (Cat #U-009200-10-05 or -20)
CRISPRmod CRISPRa Human TTN crRNA (Cat #U-009100-01-05 or -20)
CRISPRmod CRISPRa Human TTN crRNA pool (Cat #U-009100-10-05 or -20)
CRISPRmod CRISPRa Mouse Pou5f1 crRNA (Cat #U-009200-02-05 or -20)
CRISPRmod CRISPRa Mouse Pou5f1 crRNA pool (Cat #U-009200-20-05 or -20)
CRISPRmod CRISPRa Mouse Ttn crRNA (Cat #U-009100-02-05 or -20)
CRISPRmod CRISPRa Mouse Ttn crRNA pool (Cat #U-009100-20-05 or -20)
Negative control crRNA reagent:
CRISPRmod CRISPRa Non-targeting crRNA (Cat #U-009500-01-05 or -20)
CRISPRmod CRISPRa Non-targeting crRNA Pool (Cat #U-009500-10-05 or-20)
Base Medium: Appropriate antibiotic-free cell culture medium without serum
Growth Medium: Appropriate antibiotic-free cell culture medium (with serum and/or supplements) recommended
for maintenance of the cells of interest
Selection Medium: Growth Medium supplemented with the appropriate concentration of antibiotics
Additional recommended materials:
Materials for RNA isolation and quantitative RT-qPCR for gene expression analysis
Assay for assessing cell viability

Generation of stable cell line expressing dCas9-VPR
The protocol described here is designed for rapid generation of a cell population where most of the cells have single
integration of a CRISPRmod CRISPRa Lentiviral dCas9-VPR proviral sequence in the genome.
Determining blasticidin concentration for selection of transduced cells
The CRISPRmod CRISPRa Lentiviral dCas9-VPR expression vectors confer resistance to blasticidin in transduced cells.
Before transducing cells, determine the minimum concentration of blasticidin required to kill non-transduced cells(in 3 to 10 days) by generating a blasticidin kill curve. The blasticidin concentration range for many mammalian cells
is 2-15 μg/mL.
Transduction of cells with CRISPRmod CRISPRa Lentiviral dCas9-VPR expression particles
The protocol below describes 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 between cell types and must
be determined empirically for each cell line of interest.

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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 ofculture 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.

Equilibrate the Base Medium to 37 °C.

2.

Calculate the volume of lentiviral particles needed for transduction at MOI = 0.3.
The functional titer of CRISPRmod CRISPRa Lentiviral dCas9-VPR expression particles (in HEK293T cells, as
determined by qPCR) is reported on the Certificate of Analysis (C of A). We recommend an MOI ≤ 0.3 (adjusted
for relative transduction efficiency in your cell type) to ensure single integration of the lentiviral dCas9-VPR. The
relative transduction efficiency of your cell 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.

3.

Thaw the CRISPRmod CRISPRa Lentiviral dCas9-VPR 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 (if necessary) and immediately returned to -80 °C.

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4.

Once thawed, gently mix and pipette the calculated volume of lentiviral particles into 0.25 ml of the Base Medium(no serum) to create the Transduction Medium.

5.

Remove the Growth Medium from the well and add the Transduction Medium containing the lentiviral particles(see Appendix for guidelines on other plate formats).

6.

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

7.

At 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.
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 dCas9-VPR cell lines 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 experimentallyprior to selection using a kill curve. Blasticidin usually kills cells between 3 and 10 days, slow growing cells may
take longer. If the cells 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.
Record the passage number and avoid working with stable cell populations that exceed 10 passages from frozen
the stock.

Utilize the mixed population of dCas9-VPR expressing cell line obtained above for transfection with synthetic CRISPRa
guide RNAs or transduction of lentiviral CRISPRa sgRNAs for activation of 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 cells of interest.

Transfection of synthetic CRISPRmod CRISPRa guide RNAs
The following is an example protocol for delivery of CRISPRmod CRISPRa synthetic crRNA complexed with tracrRNA into
adherent U2OS cells stably expressing dCas9-VPR. The protocol is provided for transfection in 96-well plates and thevolumes are given for one well and a final 25 nM concentration of the crRNA:tracrRNA complex. We suggest performing
the transfection in triplicate wells and adjusting the volumes accordingly providing excess for the ease of pipetting.
Transfection conditions vary between cell lines and should be determined empirically. For alternative plating formats
see Table 3 for volume recommendations.
Day 1:
1.

Plate 1 × 104 U2OS-dCas9-VPR cells per well in a 96-well plate using Growth Medium.

2.

Incubate cells at 37 °C in a humidified CO2 incubator overnight.
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Optimal cell number for plating will vary with growth characteristics of specific cells and should be determined
experimentally.

Day 2:
1.

Prepare 1 μM solution of CRISPRmod CRISPRa synthetic crRNA and tracrRNA from previously prepared 10 μM stocks.

2.

In nuclease-free microcentrifuge tubes (or deep-well 96-well plates for multiple targets) prepare the synthetic
crRNA:tracrRNA complex by adding 2.5 µL of 1 µM crRNA and 2.5 µL of 1 μM tracrRNA to 5 µL of serum-free medium.

3.

Prepare a DharmaFECT transfection reagent working solution in a separate tube by diluting 0.2 µL of DharmaFECT 4
reagent in 9.8 µL of serum-free medium and mix gently. Incubate tube for 5 minutes at room temperature.
The optimal DharmaFECT Transfection Reagent formulation and concentration varies between cell lines and isaffected by the cell density. Easy-to-transfect cells and lower cell densities typically require lower amount of
DharmaFECT Transfection Reagent. For replicates, prepare sample volumes sufficient for the number of
replicates and extra to account for pipetting errors.

4.

Add 10 μL of DharmaFECT 4 working solution to each sample tube containing the crRNA:tracrRNA complex. This
brings the total volume to 20 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 80 μL antibiotic-free complete Growth Medium to each sample to bring
the total volume in each tube to 100 μL.

7.

Remove medium from the wells of the 96-well tissue culture plate with cells and replace with 100 μL of theappropriate Transfection Medium in 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 expression analysis (see Appendix).

Table 2. Suggested volumes per well for transfection of dCas9-VPR-expressing cells with 25 nM synthetic crRNA:tracrRNATube 1: crRNA:tracrRNAworking solution (µL/well)plating format well surface area
(wells/plate(cm2/well)

1 µM synthetic
tracrRNA (µL)

1 µM synthetic
crRNA (µL)

Tube 2: DharmaFECT transfection
reagent working solution (µL/well)

Base Medium(serum free) (µL)

DharmaFECT(µL)

Base Medium(serum free) (µL)

Growth Medium(µL/well)

Final transfection
volume(µL/well)

96

0.3

2.5

2.5

5

0.2

9.8

80

100

24

2

12.5

12.5

25

1

49

400

500

12

4

25

25

50

2

98

800

1000

6

10

50

50

100

5

195

1600

2000

Packaging CRISPRmod CRISPRa Lentiviral dCas9-VPR plasmids into particles
CRISPRmod CRISPRa Lentiviral dCas9-VPR expression plasmids are Tat dependent and require a packaging system that
expresses the tat gene. For packaging of lentiviral constructs, we recommend the Dharmacon Trans-Lentiviral ORF
Packaging System. For packaging protocols and additional information please consult the product manual.
CRISPRmod CRISPRa Lentiviral Blast-dCas9-VPR Plasmids do not express a fluorescent protein reporter,
therefore, after packaging of plasmid DNA, we recommend titering the lentiviral particles using a functional
lentiviral titration protocol such as limiting dilution with cell viability assay by crystal violet staining or
genomic qPCR assay.
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4

Appendix
Optimization of transfection conditions for delivery ofCRISPRmod CRISPRa synthetic
guide RNATo 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 control crRNA. The transfection
optimization can be easily performed in a 96-well format allowing 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 transfection 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 crRNA:tracrRNA complex per well

At 48 to 72 hours post-transfection, perform a cell viability assay to determine the highest lipid concentration that has
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 expression analysis to determine thecondition that produces highest gene activation. Use the determined optimal conditions for subsequent transfection ofyour selected dCas9-VPR expressing cell lines with the CRISPRmod CRISPRa synthetic guide RNA.

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

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
CRISPRmod CRISPRa Lentiviral dCas9-VPR 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.

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Plasmid DNA
CRISPRmod CRISPRa Lentiviral dCas9-VPR 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-freesolution, such as Tris buffer.
Synthetic crRNA and tracrRNACRISPRmod CRISPRa 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.

5

Frequently asked questions
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. You may
use our 10 mM Tris-HCl Buffer pH 7.4 for resuspension. We recommend that RNA oligonucleotides be resuspended toa 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 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 CRISPRmod CRISPRa 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 transfection reagent other than DharmaFECT to deliver the crRNA:tracrRNA 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 foreach cell line of interest.

What is the best way to confirm that my gene is activated?
We suggest using RT-qPCR to measure the relative change in target gene expression levels between samples treatedwith a non-targeting control and CRISPRmod CRISPRa guide RNAs. RT-qPCR analysis can be completed with either the
SYBR green method or probe-based gene expression assays. Follow manufacturer’s instructions for RNA isolation and
RT-qPCR set up and use best practices to avoid contaminations during the RNA isolation, cDNA synthesis and qPCR
set up. Use proper controls for RT-qPCR analysis (include no RNA samples, no reverse transcriptase samples, no cDNA
samples negative controls). Additionally, when performing RT-qPCR for gene activation the expression level may go fromnot detectable to expressed. In this case, when using the ∆∆Cq method of analysis, an arbitrary value representing thedetection limit of the qPCR instrument is used as a place holder for “non-detectable” as a non-zero value is necessary to
perform the calculation. In most cases this value will be between 35 and 40 depending on the number of programmed
cycles and the instrument Cq determination method. We recommend adding additional cycles (up to 45 total) to
standard qPCR cycling conditions.

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Can the CRISPRmod CRISPRa system be used for gene activation in non-mammalian organisms, such asflies or worms?
CRISPRmod CRISPRa system is designed for mammalian expression and have been tested in mammalian cells. The guide
RNAs are predesigned to activate human and mouse genes. Custom guide RNAs could be ordered that target promoter
regions of other species, however we cannot predict the efficacy of using CRISPRmod CRISPRa lentiviral dCas9-VPR
particles and CRISPRmod CRISPRa synthetic guide RNA components, nor can we troubleshoot experiments performed in
non-mammalian systems.

Can I co-transfect the CRISPRmod CRISPRa crRNA:tracrRNA components with the CRISPRmod CRISPRa
Lentiviral dCas9-VPR expression plasmid?
We do not recommend doing a co-transfection of CRISPRmod CRISPRa Lentiviral dCas9-VPR Expression plasmids withsynthetic guide RNAs due to the transient nature of transcriptional activation with the non-expressed guide RNAs and thelonger time necessary to perform blasticidin selection (typically > 5 days) to remove the non-transfected cells. For
transient expression experiments, or to avoid the use of lentiviral particles, we recommend doing a co-transfectionof the CRISPRmod CRISPRa sgRNA plasmid with the Lentiviral dCas9-VPR expression plasmid which then allows forselection of both components.
Molecule

Molecular weight

Extinction coefficient (E260)

crRNA

13,500 g / mol

423000 L/mol×cm

tracrRNA23,820 g / mol

757800 L/mol×cm

What are the average molecular weight and extinction coefficient of crRNA and tracrRNA?
Where can I obtain the CRISPRmod CRISPRa Lentiviral dCas9-VPR vector maps?
dCas9-VPR plasmid maps can be obtained upon request from Technical Support.

What is the size of the dCas9-VPR protein and what antibody do you recommend for confirmation of
expression of the dCas9-VPR?
The VPR activators add additional 536 amino acids to dCas9 which shift the molecular weight of the dCas9-VPR toapproximately ~220 kDa. The protein could be detected using Cas9 antibodies (for example: Novus Biologicals
cat #NBP2-36440).

Can I use the CRISPRmod CRISPRa guide RNA predesigned for gene knockout in CRISPRa experiments?
The guide RNA designs for CRISPRa are different than the CRISPRmod CRISPRa guide RNAs for CRISPR-Cas9 knockout
experiments. The guide RNA designs for CRISPRa are required to bind upstream of the transcriptional start site and are
based on a published CRISPRa algorithm. Pre-designed CRISPRmod CRISPRa guide RNAs are optimized for functional geneknockout with the Dharmacon algorithm and target the gene’s coding region.

What if a gene has more than one transcriptional start site?
The published CRISPRa v2 algorithm (Horlbeck et al., 2016) used FANTOM and Ensembl databases to predict moreaccurately the transcriptional start site (TSS). Some genes (6.8%) were identified as having alternative transcriptional startsites. The publication lists 10 gRNA designs per TSS. For the CRISPRmod CRISPRa predesigned guide RNAs, we offer the top
four guide RNAs for the primary TSS, and, when applicable, four guide RNAs for the secondary TSS. These are labeled as P1
and P2, respectively. If the CRISPRmod CRISPRa guide RNAs for your gene do not have a P2 designation, only a single startsite is designated for that gene. If your gene has both P1 and P2 guide RNAs, it might be beneficial to test both in yourexperiment as which TSS is active and to what level depends on your cell line. For a small number of genes (0.1%)
Horlbeck et al., 2016 identified more than two TSS. We only offer P1 and P2 designs as catalog items, but we can
13generate the additional guide RNAs as a custom request based on designs from the published algorithm.

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Technical Manual

How specific are the gRNAs in targeting the gene of interest?
Several publications have shown CRISPRa to be highly specific by RNA seq expression analysis, but CRISPRa is a newtechnology and off-targeting still needs to be explored in more detail. Keep in mind that for CRISPRa off-targeting, theguide RNA needs to bind to the promoter region of another gene in order to have an off-target effect, which dramaticallydecreases the potential off-target space. Furthermore, the guide RNAs are designed based on a published algorithm thatincorporates chromatin, nucleosome position, and sequence features to accurately predict highly effective guide RNAs
and also applies a filter for off-target binding.
However, there might be examples of genes where the promoter region for one gene is in close proximity to anothergene’s promoter region. Investigation of the genomic location for your gene of interest and performing expression
analysis to confirm activation of the target gene without having effects on other proximal genes might be important for
proper interpretation of the phenotypic analysis.

Can I use the CRISPRmod CRISPRa guide RNAs with the SunTag system?
Yes. The CRISPRmod CRISPRa system uses canonical guide RNAs and they can be used with other similar systems that use
canonical guide RNAs, like the SunTag system.

Can I use CRISPRmod CRISPRa crRNA in the dCas9-SAM system?
Yes. Synthetic CRISPRmod CRISPRa crRNA may be used with synthetic CRISPRmod MS2 tracrRNA for activation in cells
expressing the dCas9-SAM system.

What level of overexpression should I expect?
The level of gene activation correlates with the basal expression of the gene, and is therefore cell-line specific. For genesthat are either not expressed or expressed at a low level, we generally see high activation (100 to 10,000 fold over NTC
treated cells). But for genes that are expressed at a high level, CRISPRa causes lower activation (2 -100 fold). Therefore,knowing the level of expression of the gene of interest in your cells can help you gauge the expectation for the levelof activation.

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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.

Qi, L.S., et al., Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.
Cell, 152(5): p. 1173-83 (2013)

4.

L.A.Gilbert, et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 154(2):p. 442-51 (2013).

5.

A.W. Cheng, et al., Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional
activator system. Cell Res, 23(10): p. 1163-71 (2013).

6.

A. Chavez et al., Highly efficient Cas9-mediated transcriptional programming. Nat. Methods. 12, 326–328 (2015).

7.

M. A. Horlbeck et al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression andactivation. eLife. 5, e19760 (2016).

8.

L. A. Gilbert et al., Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 159, 647–661(2014).

9.

M. E. Tanenbaum, L. A. Gilbert, L. S. Qi, J. S. Weissman, R. D. Vale, A protein-tagging system for signal amplification ingene expression and fluorescence imaging. Cell. 159, 635–646 (2014).

10. S. Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 517,
583–588 (2015).

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Lentiviral particle product safety level information
This lentiviral particle product safety level information constitutes Product Documentation according to clause 1 of 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 their local
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 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
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

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In the US:
For US guidance on containment for lentiviral vectors, please refer to:
1.
2.
3.

The Recombinant DNA Advisory Committee (RAC) guidelines for research with lentiviral vectors.
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);
The NIH Guidelines For Research Involving Recombinant DNA Molecules

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

2.

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); andCouncil 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.

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 theabovementioned 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.

8

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

Limited use licenses
The gene editing 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 particularapplication. Please review the Label Licenses governing all use of the Products.

To find the contact information in your country for your technology of interest, please visit us at
horizondiscovery.com/contact-us
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom©2021 The Horizon logo and other trademarks are the property of Horizon Discovery Limited, unless otherwise stated. DHARMACON and EDIT-R are trademarks of Dharmacon Inc.

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