Cas9 driven by an optimal promoter improves gene editing ineukaryotic cell lines when paired with synthetic crRNA and tracrRNAAmanda Haupt, Emily Anderson, Žaklina Strezoska, Hidevaldo Machado, Shawn McClelland, Maren Mayer, Adam Rocker, Annaleen Vermeulen, Amanda Birmingham,Melissa Kelley, Anja Smith
Dharmacon, 2650 Crescent Drive, Suite #100, Lafayette, CO 80026, US

Expression of Cas9 by stronger promoters yields higher percentages of geneknockout in the same cell line when multiple promoters are compared. Inmouse embryonic stem cells, Cas9 expression driven by mEF1α, followed by
the hEF1α, showed higher frequency of mutations (indel) compared to themore commonly used hCMV promoter.
Mouse ES-D3 cells

A.

hCMVmCMV

Cell populations were selected with puromycin for 48 h and an increase in thefrequency of detected indels was observed in A549 and U2OS cells compared
to unselected populations (Figure 5).
A. A549

B. U2OShEF1α

CDKN1AUn
transfecteUndse
lec
tedSe
lectedMW(bp)Interest in genome engineering of mammalian cells has been increasing inthe past few years with the development of new tools to create DNA breaks atspecific locations on the cell genome. Among these tools, the CRISPR (clusteredregularly interspaced short palindromic repeats)-Cas9 (CRISPR associatedprotein 9) system has gained significant interest due to its relative simplicity
and ease of use compared to other genome engineering technologies. TheCRISPR-Cas9 system requires a complex of the Cas9 protein with a transactivating RNA (tracrRNA) and a gene-targeting CRISPR RNA (crRNA) (Figure 1),or a single guide RNA (sgRNA, a chimeric form of tracrRNA with a crRNA).

Enrichment of edited cells by antibiotic selection

Untransfec
UntedselectedSe
lectedIntroduction

Untransfec
teUndselectedSe
lectedMW(bp)

Optimal promoter for Cas9 expression improvesgene editing

PPIB

EMX1
1500

mEF1α

PGK

1500

CAG

850

850

PAM

Genomic DNA

400
400
200
200

5'tracrRNA3'

MW (bp)

Untransf.

CAG

PGK

mEF1α

hEF1α

hCMV

mCMV

Ppib

Untransf.

CAG

PGK

mEF1α

3'hEF1α5'mCMV

crRNA

hCMVB.

Dnmt3bIndel (%) 0

Mismatched inhibitor target sites show that
position of mismatch affects functionality
1500

850
400200Figure 1. Gene editing with a three-componentSite-specificsystem: Cas9 nuclease (lightblue), programmed by the tracrRNA:crRNA complex (blue and green, respectively)
dsDNA breakcutting both strands of genomic DNA 5' of the PAM (red). A synthetic approach totracrRNA:crRNA complex enables fast assessment of multiple target sites per gene orPAM
for multiple genes without the requirement of any cloning steps.

Indel (%):

26722315612FACS or antibiotic selection can be used to obtain cell populations withincreased likelihood of Cas9-induced gene engineering events. We observed
up to two-fold increase of mutations in a population of HEK293T or U2OS cells
after enrichment by FACS compared with unsorted cells after transient coStrand withinvasiontransfection
the hCMV::mKate2-Cas9 plasmid and chemically synthesizedtracrRNA
crRNA targeting PPIB (Figure 4).
DNAandsynthesis
A.

A.

B.

HEK293T
NegativeLow

MediumHighB.

ted nfec ectio ivesmnlt
diu ightra o se ega owUnHMeNNL

Before selection

After selection

MW (bp)% GatedThe strength of commonly used constitutive mammalian promoters varies
among different cell types and cellular contexts. It has been previously
shown that the likelihood of a gene editing event is dependent on thenuclease expression level (Certo et al., 2011, Fu et al., 2013). Therefore, toachieve efficient gene knockout one must assess the most suitable promoter
to express Cas9 in the cell line of choice. In addition, the ability to enrich forcells transfected with CRISPR-Cas9 system may be advantageous for one
particular experimental condition. To evaluate the effect of promoter
Precise choicemodification
on expression of Cas9 in multiple cell lines we constructed twelve differentvectors expressing Cas9 from six promoters allowing for enrichment either by
antibiotic selection or FACS analysis (Figure 2).

21001230

Figure 5. PuroR-Cas9 bicistronic plasmids allow enrichment of gene edited cells
by quick selection with puromycin. A549 and U2OS cells were transientlyRR
co-transfectedwithPuro-Cas9 expressing plasmids, synthetic crRNA andFigure 5. Puro -Cas9 bicistronic plasmids allow enrichment of genetracrRNA
described
in Figure
3. Cells weretreatedwith (A)1.5 or (B) 2 μg/mL
editedcells by asquick
selectionwith puromycin.
U2OScells weretransientlyR
puromycinwith
24 hPuro
post-transfection
andplasmids,
selectedsynthetic
for 48 h. crRNA
Percentage
co-transfected-Cas9 expressing
and of mutations(indel)determinedby3.mismatchassay
tracrRNAasweredescribed
in Figure
Cells weredetection
treated with(A) (T7Endonuclease
1.5 or (B) 2 µg/mL I, NEB) andpuromycin
24 h post-transfection
selected for
48 h. Percentage
ofcalculated
by densitometry andas describedin Figure3.

Conclusion• Utilizing a highly active promoter for Cas9 expression enables better editingin specific cell lines.

• Efficient gene editing can be achieved with a three-component system:
plasmid Cas9 and synthetic tracrRNA and crRNAs.

DonorDNA

Optimal promoter for Cas9 expression improvesgene editing0• Enrichment of transiently transfected cells either by FACS or puromycin
selection can further improve the yield of edited cells.

Enrichment of edited cells by FACS

Deletions17mutations (indel) were determined by mismatch detection assay (T7Endonuclease I,NEB) and calculated by densitometry as previously described (Cong et al., 2013).

Figure 3. mKate2-Cas9 expression from different promoters in mouse ES-D3
cells. mEF1α drives the best Cas9 expression and gene editing in the murine
embryonic cell line ES-D3. Cells were transiently co-transfected with mKate2Cas9 expressing plasmids, synthetic tracrRNA and crRNA using Dharmacon
Dharmafect™ Duo as the transfection reagent. A. Cells were imaged at48 h post-transfection with 20x magnification, 2 s exposure and 2 Gain.
B. Percentage of mutations (indel) were determined by mismatch detection
assay (SURVEYOR™, Transgenomic) and calculated by densitometry as
previously described (Cong et al., 2013).

Presented here are results on the efficiency of using synthetic crRNA andHDR
tracrRNA to introduce geneNHEJ
editing events whenco-transfected with aplasmid expressing Cas9. We explored the use of antibiotic and fluorescenceactivated cell sorting (FACS) methods for enrichment of cells that haveundergone gene editing, and the use of multiple promoters to increase
efficiency of gene editing with Cas9 and synthetic tracrRNA and crRNAs.

• Use of synthetic tracrRNA and crRNAs is a simplified method for gene
editing of one or more genes without requiring any cloning steps.
• By virtue of its simplicity, this three-component CRISPR-Cas9 system isamenable to high-throughput genome editing applications.

References

1500850

A

400

1. M. T. Certo et al., Tracking genome engineering outcome at individual DNA
breakpoints. Nat Methods 8, 671-676 (2011).

200Figure 2 (Smith)Indel (%)

041430242. Y. Fu et al., High-frequency off-target mutagenesis induced by CRISPR-Cas
nucleases in human cells. Nat. Biotechnol. 31, 822-826 (2013).

27Log Fluorescence Intensity
hCMV::mKate2-Cas9 + tracrRNA:crRNA-PPIB

3. L. Cong, et al., Multiplex genome engineering using CRISPR/Cas systems.
Science 339, 819-823 (2013).

Untransfected control

A.A.

B.
B.

hCMV
mCMV
hEF1α
mEF1α
PGK
CAG
mKate2 T2A Cas9pA

hCMV
mCMV
hEF1α
mEF1α
PGK
CAG

C. C.

U2OS
NegativeLow

High

D.
D.

tedcsfentraUnNo

oncti

lese

Before selection

etivNaeghw

Lo

After selection

Hig% GatedInsertions

31MW (bp)

1500
850

400

PuroRT2A Cas9pA

200

Indel (%)

01132024

Log Fluorescence Intensity
hCMV::mKate2-Cas9 + tracrRNA:crRNA-PPIB
Untransfected control

Figure 2. Plasmid variants for Cas9 expression under control of a collectionof constitutive promoters. Cas9 protein is expressed as a bicistronic transcript
using a 2A “self-cleaving” peptide linking Cas9 to either (A) mKate2 (far-red
fluorescent protein reporter) or (B) the puromycin resistance gene (PuroR)
followed by the bovine growth hormone polyadenylation signal (pA).

Figure 4. mKate2-Cas9 bicistronic plasmids allow enrichment of gene edited cellsby FACS. HEK293T and U2OS cells were transiently co-transfected with mKate2-Cas9
expressing plasmids and synthetic crRNA and tracrRNA as described previously. Cells
were sorted 72 h post-transfection. Percentage indel for the PPIB gene target wasdetermined as described in Figure 3. Cells populations were sorted by their relative
red fluorescence intensity as background (negative), low, medium or high.

horizondiscovery.com/dharmacon
For more information:
If you have any questions directly related to this document, contact our experts:
UK +44 (0) 1223 976 000
USA +1 800 235 9880, +1 303 604 9499
Website horizondiscovery.com/contact-us