Cas9 driven by an optimal promoter improves gene editing in

eukaryotic cell lines when paired with synthetic crRNA and tracrRNA
Amanda Haupt, Emily Anderson, Žaklina Strezoska, Hidevaldo Machado, Shawn McClelland, Maren Mayer, Adam Rocker, Annaleen Vermeulen, Amanda Birmingham,
Melissa Kelley, Anja Smith
Dharmacon, now part of GE Healthcare, 2650 Crescent Drive, Suite #100, Lafayette, CO 80026, US

Optimal promoter for Cas9 expression improves

Introduction Enrichment of edited cells by antibiotic selection
gene editing

Interest in genome engineering of mammalian cells has been increasing in Expression of Cas9 by stronger promoters yields higher percentages of gene Cell populations were selected with puromycin for 48 h and an increase in the
the past few years with the development of new tools to create DNA breaks at knockout in the same cell line when multiple promoters are compared. In frequency of detected indels was observed in A549 and U2OS cells compared
specific locations on the cell genome. Among these tools, the CRISPR (clustered mouse embryonic stem cells, Cas9 expression driven by mEF1α, followed by to unselected populations (Figure 5).
regularly interspaced short palindromic repeats)-Cas9 (CRISPR associated the hEF1α, showed higher frequency of mutations (indel) compared to the
protein 9) system has gained significant interest due to its relative simplicity more commonly used hCMV promoter.
and ease of use compared to other genome engineering technologies. The A. A549 B. U2OS

d
CRISPR-Cas9 system requires a complex of the Cas9 protein with a trans- Mouse ES-D3 cells

te

te

te
d

d
fec

fec

fec
te

te

te
activating RNA (tracrRNA) and a gene-targeting CRISPR RNA (crRNA) (Figure 1), A.

d
hCMV mCMV hEF1α

)
ns

ns

ns
lec

lec

lec
(bp

(bp
te

te

te
tra

tra

tra
lec

lec

lec
or a single guide RNA (sgRNA, a chimeric form of tracrRNA with a crRNA).

se

se

se
MW

MW
Un

Un

Un

Un

Un

Un
Se

Se

Se
CDKN1A PPIB EMX1
1500
1500
mEF1α PGK CAG 850
850

Genomic DNA PAM 400

400

200
200

Dnmt3b Ppib

Untransf.

Untransf.
B.

MW (bp)
Indel (%) 0 1 17 0 2 10 0 12 30

mEF1α

mEF1α
mCMV

mCMV
hEF1α

hEF1α
hCMV

hCMV
CAG

CAG
PGK

PGK
crRNA 5' 3'
Figure 5. PuroR-Cas9 bicistronic plasmids allow enrichment of gene edited cells
5' Mismatched inhibitor target sites show that by quick selection with puromycin. A549 and U2OS cells were transiently

tracrRNA position of mismatch affects functionality 1500 co-transfected

R with PuroR
-Cas9 expressing plasmids, synthetic crRNA and
Figure 5. Puro -Cas9 bicistronic plasmids allow enrichment of gene
tracrRNA
3'
850
edited cells by as described
quick in Figure
selection 3. Cells were
with puromycin. treated
U2OS with (A)
cells were 1.5 or (B) 2 μg/mL
transiently
puromycinwith
co-transfected 24 hPuro
post-transfection
R
-Cas9 expressingandplasmids,
selectedsynthetic
for 48 h. crRNA
Percentage
and of mutations
400
(indel)
tracrRNA aswere determined
described by3.mismatch
in Figure Cells weredetection assay
treated with (A) (T7Endonuclease
1.5 or (B) 2 µg/mL I, NEB) and
200 calculated
puromycin by densitometry and
24 h post-transfection as described in Figure
selected for 3.
48 h. Percentage of
mutations (indel) were determined by mismatch detection assay (T7Endonuclease I,
Figure 1. Gene editing with a three-componentSite-specific
system: Cas9 nuclease (light Indel (%): 3 2 6 7 2 2 3 1 5 6 1 2
NEB) and calculated by densitometry as previously described (Cong et al., 2013).
blue), programmed by the tracrRNA:crRNA complex (blue and green, respectively)
dsDNA break
cutting both strands of genomic DNA 5' of the PAM (red). A synthetic approach to
Figure 3. mKate2-Cas9 expression from different promoters in mouse ES-D3
tracrRNA:crRNA complex enables fast assessment of multiple target sites per gene or
PAM
for multiple genes without the requirement of any cloning steps.
cells. mEF1α drives the best Cas9 expression and gene editing in the murine
embryonic cell line ES-D3. Cells were transiently co-transfected with mKate2- Conclusion
Cas9 expressing plasmids, synthetic tracrRNA and crRNA using Dharmacon
Dharmafect™ Duo as the transfection reagent. A. Cells were imaged at
48 h post-transfection with 20x magnification, 2 s exposure and 2 Gain.
Presented here are results on the efficiency of using synthetic crRNA and
tracrRNA to introduce geneNHEJ HDR B. Percentage of mutations (indel) were determined by mismatch detection
editing events when co-transfected with a assay (SURVEYOR™, Transgenomic) and calculated by densitometry as
• Utilizing a highly active promoter for Cas9 expression enables better editing
plasmid expressing Cas9. We explored the use of antibiotic and fluorescence- previously described (Cong et al., 2013). in specific cell lines.
activated cell sorting (FACS) methods for enrichment of cells that have
undergone gene editing, and the use of multiple promoters to increase • Enrichment of transiently transfected cells either by FACS or puromycin
efficiency of gene editing with Cas9 and synthetic tracrRNA and crRNAs. selection can further improve the yield of edited cells.
• Efficient gene editing can be achieved with a three-component system:
Deletions Enrichment of edited cells by FACS plasmid Cas9 and synthetic tracrRNA and crRNAs.
Donor
• Use of synthetic tracrRNA and crRNAs is a simplified method for gene
DNA editing of one or more genes without requiring any cloning steps.
FACS or antibiotic selection can be used to obtain cell populations with
Optimal promoter for Cas9 expression improves increased likelihood of Cas9-induced gene engineering events. We observed • By virtue of its simplicity, this three-component CRISPR-Cas9 system is
Insertions gene editing up to two-fold increase of mutations in a population of HEK293T or U2OS cells amenable to high-throughput genome editing applications.
after enrichment by FACS compared with unsorted cells after transient co-
Strand with
transfection invasion
the hCMV::mKate2-Cas9 plasmid and chemically synthesized
The strength of commonly used constitutive mammalian promoters varies DNAand
tracrRNA synthesis
crRNA targeting PPIB (Figure 4).
among different cell types and cellular contexts. It has been previously A. B.
shown that the likelihood of a gene editing event is dependent on the A. Negative Low
HEK293T
Medium High B. n s
ted n
fec ectio ive
l t m
tra o se ega ow diu igh

References
Un Me
nuclease expression level (Certo et al., 2011, Fu et al., 2013). Therefore, to
N N L H
Before selection After selection MW (bp)

achieve efficient gene knockout one must assess the most suitable promoter
% Gated

to express Cas9 in the cell line of choice. In addition, the ability to enrich for 1500

cells transfected with CRISPR-Cas9 system may be advantageous for one 850

particular experimental condition. To evaluate the effect of promoter

Precise choice
modification A 400
1. M. T. Certo et al., Tracking genome engineering outcome at individual DNA
on expression of Cas9 in multiple cell lines we constructed twelve different breakpoints. Nat Methods 8, 671-676 (2011).
200
vectors expressing Cas9 from six promoters allowing for enrichment either by
Figure 2 (Smith)
2. Y. Fu et al., High-frequency off-target mutagenesis induced by CRISPR-Cas
antibiotic selection or FACS analysis (Figure 2).
Indel (%) 0 14 4 24 30 27
Log Fluorescence Intensity
nucleases in human cells. Nat. Biotechnol. 31, 822-826 (2013).
hCMV::mKate2-Cas9 + tracrRNA:crRNA-PPIB
Untransfected control
3. L. Cong, et al., Multiplex genome engineering using CRISPR/Cas systems.
A.A. B.
B. Science 339, 819-823 (2013).
hCMV hCMV C. C. U2OS D.
D.
sfe
cted
cti
on e
Negative Low High n le tiv
mCMV mCMV Un
tra
No
Before selection
se
N eg
a
Lo
w
After selection
Hig
h
MW (bp)

hEF1α hEF1α
mEF1α mEF1α gelifesciences.com/dharmacon
% Gated

1500

PGK PGK 850

CAG CAG 400

mKate2 T2A Cas9 pA Puro T2A Cas9 pA

200
R
Indel (%) 0 11 3 20 24
Log Fluorescence Intensity
hCMV::mKate2-Cas9 + tracrRNA:crRNA-PPIB
Untransfected control

Figure 2. Plasmid variants for Cas9 expression under control of a collection Figure 4. mKate2-Cas9 bicistronic plasmids allow enrichment of gene edited cells
of constitutive promoters. Cas9 protein is expressed as a bicistronic transcript by FACS. HEK293T and U2OS cells were transiently co-transfected with mKate2-Cas9
using a 2A “self-cleaving” peptide linking Cas9 to either (A) mKate2 (far-red expressing plasmids and synthetic crRNA and tracrRNA as described previously. Cells
fluorescent protein reporter) or (B) the puromycin resistance gene (PuroR) were sorted 72 h post-transfection. Percentage indel for the PPIB gene target was TM
followed by the bovine growth hormone polyadenylation signal (pA). determined as described in Figure 3. Cells populations were sorted by their relative
red fluorescence intensity as background (negative), low, medium or high.