A novel fluorescent surfactant enhances the delivery of the Cas9 ribonucleoprotein and enables the identification of edited cells

Article abstract of DOI:10.1039/c9cc00261h

In this report, we designed and synthesized a novel fluorescent single tailed surfactant (termed FEDS), which can disrupt endosomes, complex lipofectamine, and can also identify cells that have been transfected. FEDS was able to increase the gene editing

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
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A novel fluorescent surfactant enhances the delivery of
the Cas9 ribonucleoprotein and enables the
identification of edited cells
Cite this: DOI: 10.1039/x0xx00000x
Jie Li‡a, Joachim Justad Røise‡ a, Jingtuo Zhang ‡ a, Jiaying Yang a, D. Lucas Kerr a,
Hesong Han a and Niren Murthy* a
Received 00th January 2015,
Accepted 00th January 2015
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this report, we designed and synthesized a novel fluorescent
single tailed surfactant (termed FEDS), which can disrupt
endosomes, complex lipofectamine, and can also identify cells that
have been transfected. FEDS was able to increase the gene editing
efficiency of lipofectamine/Cas9 ribonucleoprotein by 300% via a
combination of fluorescent based enrichment and endosomal
disruption.
Therapeutics based on proteins, RNA and DNA have
tremendous therapeutic potential but have been challenging to
develop due to delivery problems.1–3
Delivering such
biomacromolecules is challenging because it requires
developing multifunctional drug delivery vehicles that can
perform multiple functions, in particular, biomolecule
complexation, endosomal disruption, and also imaging of cells
that have been transfected.4–6 Although a wide number of
peptides, proteins and polymers have been developed that can
disrupt endosomes,7–11 and a wide number of dyes, proteins
and quantum dots have been developed that can image
transfected cells,12–14 combining imaging agents with
endosomal disruptive agents together into a single delivery
vehicle remains a challenge because of their large size and
molecular complexity.6 There is therefore a great need for the
development of molecular scaffolds with simple and well
defined chemical structures that can efficiently incorporate
multiple functions into a single delivery vehicle.
Single tailed surfactants based on Triton-X have great
potential to serve as scaffolds for developing multifunctional
drug delivery vehicles. Triton-X based surfactants have a low
molecular weight (<1,000),15 yet have a membrane disruptive
efficiency comparable to peptide based endosomal disruptive
agents,16 which are 5-10 times larger in molecular weight. In
addition, Triton-X based surfactants are straightforward to
synthesize and non-antigenic, as they are composed of PEG
chains conjugated to alkyl chains. More importantly, Triton-X
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pH of the endosome FEDS should recover its membrane
disruptive ability because of protonation of its carboxyl group
(See Fig. 1b). In addition, the negatively charged carboxyl group
of FEDS should allow it to assemble with cationic lipid/
biomolecule complexes to form FEDEX. Finally, the methyl-
amino-benzyl amide fluorophore in FEDS will identify cells that
have internalized FEDEX, and this can be used to enrich for cells
that have been transfected. For example, in this report we used
the fluorescence of FEDS to enrich for cells that were gene
edited, after transfection with FEDEX (See Fig. 1b).
FEDS was synthesized according to the scheme shown in Fig.
2a and was synthesized in 6 steps. Firstly, the hydrophobic
domain of FEDS was constructed by alkylating isatoic anhydride
with 1-bromo-8-tritylmercaptooctane (1), using sodium hydride
as a base. The hydrophilic portion of FEDS was formed by
performing a ring-opening reaction on the anhydride ring (2)
with amino-PEG (1000 Mw). The thiol group of FEDS was then
deprotected, and the final compound was synthesized by
performing a disulfide exchange reaction between compound 4
Fig. 1 Fluorescent endosomal disruptive complex (FEDEX)
enhances the delivery of the Cas9 ribonucleoprotein in cells and
enables the identification of edited cells. a) Structure of FEDS,
FEDS contains a PEG chain, a fluorophore, an aliphatic chain,
and a carboxylate connected by a disulfide linker. b) FEDS is
incorporated into lipofectamine based liposomes that contain
Cas9 RNP to form FEDEX. Cells that internalize FEDEX can be
identified by flow cytometry due to the fluorescence of FEDS,
and this enriches for cells that have been edited. In addition,
FEDS can disrupt endosomes, and this further increases the
gene editing efficiency of the Cas9 RNP/lipofectamine
complex.
based surfactants can potentially be engineered to incorporate
other functional moieties, such as a fluorophore, without losing
their membrane disruptive activity. This is because Triton-X
based surfactants disrupt membranes due to their cone shaped
molecular structure, and will tolerate modifications as long as
their cone shaped structure is maintained.17 However, the
ability of Triton X based surfactants to act as multifunctional
delivery scaffolds has never been investigated.
In this report, we designed and synthesized a fluorescent
endosomal disruptive surfactant termed FEDS, which can
disrupt endosomes, complex cationic lipid based delivery
vehicles, and can also identify cells that have been transfected
with cationic lipids. FEDS is composed of a methyl-amino-benzyl
amide fluorophore, connected to a 10 carbon alkyl chain, and
also has hydrophilic PEG and carboxyl groups at its termini (See
Fig. 1a). FEDS has an amphiphilic structure similar to Triton X,
and consequently should be membrane disruptive. In addition,
FEDS has its hydrophobic alkyl group terminated with a
hydrophilic carboxyl group, which is designed to prevent it from
being membrane disruptive at pH 7.4. However, at the acidic
and 3-(pyridin-2-yldisulfaneyl)propanoic acid. FEDS has an
excitation maximum at 350 nm and an emission maximum at
460 nm (Fig. 2b). The quantum yield of FEDS was also
measured,18,19 using quinine sulfate in 0.05M H2SO4 as a
standard, and had a quantum yield of 0.15 (Fig. S1).
FEDS is designed to enhance the transfection ability of
lipofectamine by disrupting endosomes. For this to be possible,
FEDS has to selectively disrupt endosomal membranes over the
cellular plasma membrane. To probe for this ability, a hemolysis
assay was used to determine if FEDS has pH sensitive membrane
disruptive properties. The hemolysis assay was performed at
pH 5.5, which mimics the pH of the endosome, and at pH 7.4,
which mimics the extracellular environment. FEDS was
incubated with rabbit erythrocytes for 30 minutes at 37°C, and
the samples were centrifuged and the hemoglobin release,
O
O
O
a)
Ph
N
H
O
, NaH
(Ph) CSH, K CO
O
3
2
3
Ph
Br
Br
Br
S
Ph
DMF
48%
N
O
MeCN
41%
Ph
S
Ph
Ph
1
2
O
O
H2N-PEG1000-OMe,
TEA
O
O
TFA,Et SiH
3
N
H
N
n
n
H
SH
DCM
74%
DMF
93%
NH
N
H
Ph
S
Ph
4
Ph
3
b)
Absorption
Emission
O
N
S
S
OH
O
(5)
AcOH
O
O
N
n
H
S
OH
S
N
H
DCM
87%
O
FEDS
300
400
500
600
Wavelength (nm)
Fig. 2 Synthesis of FEDS and its fluorescent properties. a) Six step synthetic route used to generate FEDS. Briefly, isatoic
anhydride was alkylated with 1-bromo-8-tritylmercaptooctane, a ring-opening reaction was performed with the anhydride
ring with amino-PEG to form compound 3. The final FEDS was synthesized by performing a disulfide exchange reaction
between compound 3 and compound 4. b) Fluorescent spectrum of FEDS with excitation at 350 nm.
2 | Chem. Commun., 2015, 00, 1-3
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triggered by membrane disruption, was determined by sequence in Ai9 myoblasts is knocked out by gene editing, then
measuring the absorbance at 541nm. The result, shown in Fig. Ai9 myoblasts express tdTomato, which can be detected by flow
3, illustrates that FEDS caused hemolysis of rabbit RBCs at pH cytometry. We found that Ai9 myoblasts treated with
5.5 but not at pH 7.4. We performed a hemolysis study with lipofectamine 2000+ Cas9 RNP were 1.41% tdTomato positive,
FEDS as well as its reduced precursor 4. The reduced form of whereas FEDEX treated cells were 2.47% tdTomato positive,
FEDS should be membrane disruptive at both pH 5.5 and 7.4, which represents a 75% increase in editing efficiency (Fig. S4).
because of the lack of a carboxylic acid group. Fig. 3b These results show that FEDS can enhance the intracellular
demonstrates that reduced FEDS is membrane disruptive at delivery of biomolecules.
both pH 5.5 and 7.4. This suggests that the pH-dependent
membrane disruptive nature of FEDS is primarily caused by its
carboxylic acid group. FEDS therefore has the pH dependency
needed to function as an endosomal disruptive agent.
O
O
a)
b)
O
O
80
70
60
50
40
30
20
10
0
O
NH
80
70
60
50
40
30
20
10
0
n
NH
n
S
SH
N
S
OH
N
H
H
0
2
4
6
8
10
0
2
4
6
8
10
Concentration (mg/mL)
Concentration (mg/mL)
n
Fig. 3 Hemolysis efficiency of FEDS at pH 5.5 ( ) and pH 7.4
( ) at different concentrations. a) FEDS caused strong
u
hemolysis at pH 5.5, but no hemolysis at pH 7.4. b) The
reduced FEDS is membrane disruptive at both pH 5.5 and pH
7.4. The absorbance at 541 nm of rabbit RBCs incubated in
water followed by three freeze-thaw cycles was set as the
100% hemolysis standard.
Fig. 4 FEDS enhances the ability of lipofectamine to deliver
the Cas9 RNP. Knockdown of GFP in RT-HEK cells treated
with different Cas9 RNP formulations. FEDS dramatically
improved the ability of lipofectamine 2000 (FEDEX vs Lipo
2000/RNP) to deliver the Cas9 RNP.
FEDS is designed to enhance the intracellular delivery of
biomolecules by disrupting endosomes. We investigated if
FEDS could enhance the ability of lipofectamine to deliver Cas9
RNP by forming FEDEX. GFP-expressing RT HEK cells, under the
control of a doxycycline (Dox) promoter, and Cas9 RNP designed
to knock out the GFP gene via indel mutations were used for
these experiments. Cas 9 RNP was formed by first annealing
crRNA with tracrRNA at a 1:1 molar ratio at 25 µM and was
further assembled with Cas9 protein at a 1.1/1 (RNA/protein)
molar ratio on ice for 10min. FEDEX was formed by first
assembling Cas9 RNP and lipofectamine 2000 at a 5 µL (Cas9
protein 1µg/µL) to 1µL ratio, this complex was then mixed with
a 100 µg of FEDS in 150 µL of Opti-MEM medium. RT HEK cells
were transfected with FEDEX for 24 hours and further activated
by Dox to determine the percentage of GFP negative cells, via
flow cytometry. Gene editing efficiency was calculated by
subtracting the percentage of GFP negative cells in the
untreated cells from the percentage of GFP negative cells in the
treated cells (See Fig. S3). Fig. 4 demonstrates that FEDS
dramatically improved the ability of lipofectamine 2000 to
deliver the Cas9 RNP. RT HEK cells treated with lipofectamine
2000 were 13.2% GFP negative, whereas RT HEK cells treated
with FEDEX were 24.2% GFP negative. In addition, this
improvement in transfection activity was further verified via the
T7E1 endonuclease assay (Fig. S7).
FEDS has a fluorophore embedded in it, and hence can be
used to identify cells that have internalized high amounts of it.
The level of FEDEX internalization was hypothesized to correlate
with the rate of gene editing, because cells that have
internalized high amounts of FEDEX should also have
internalized high amounts of Cas9 RNP. Enriching for cells that
have a high rate of gene editing has numerous potential
applications in biology (Fig. 5a). At present, for most cell lines,
gene editing can only be determined by isolating cell clones and
performing deep sequencing. This requires a significant amount
of time and investment.21,22
We therefore performed experiments to determine if FEDS
can enrich for cells that have been gene edited. RT HEK cells
were incubated with FEDEX, and the cells were then sorted by
FACs to identify cells that had internalized high and low
amounts of FEDEX, and their levels of gene editing were
compared. As a control, cells were incubated with Cas9 RNP
and lipofectamine 2000.
Overall, cells treated with
Lipo2000/RNP had a moderate GFP knockdown rate of 16.7%
(Fig. 5d, Fig. S8a) and cells with low levels of FEDEX internalized
had a GFP knockdown rate of 20.6% (Fig. 5a, Fig. S8b). In
contract, cells with high levels of FEDEX had a striking increase
in gene editing, and had a GFP knockdown rate of 50.9% (Fig.
5b, Fig. S8b). These results demonstrate that FEDEX can enrich
for cells that have been gene edited. The fluorescence of FEDEX
can also be visualized by fluorescence microscopy, and
fluorescent microscopy can also be potentially be used to enrich
for gene edited cells (See supporting information Fig. S6).
We further tested if FEDEX could enhance gene editing in
primary Ai9 myoblasts, which are a challenging to transfect cell
preparation. Ai9 myoblasts have the tdTomato gene containing
a STOP cassette integrated into their genome.20 If the STOP
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In conclusion, we present here a new fluorescent surfactant R01AI117064, R33AI119115, R01EB020008) and the Innovative
FEDS, which enhances the ability of lipofectamine to deliver the Genomics Institute.
Cas9 RNP. In addition, FEDEX is fluorescent and cells that have
internalized high amount of FEDEX can be identified via flow
Conflicts of interest
There is no conflict of interests to declare.
cytometry, and this enables the enrichment of cells that have
been gene edited. We anticipate numerous applications of
FEDS, given its unique combination of fluorescence and
endosomal disruptive ability.
Notes and reference
a Department of Bioengineering, University of California, Berkeley,
Berkeley, CA 94720, USA. Email: nmurthy@berkeley.edu
† Electronic Supplementary Information (ESI) available: See DOI:
10.1039/c000000x/
‡ These authors contributed equally to this work.
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Fig. 5 FEDEX can enrich for cells that have been gene
edited. a) Schematic illustration of FACS sorting and
enrichment of edited cells. Cells that have internalized large
amounts of FEDEX can be identified by flow cytometry.
These cells have a higher rate of gene editing, and FEDEX
enables the enrichment of gene edited cells. b) GFP
expression of RT HEK cells sorted from FEDEX negative cells
after Dox activation. c) GFP expression of FEDEX positive RT
HEK cells after Dox activation. d) GFP expression of RT HEK
cells transfected by lipo2000/RNP after Dox activation. e)
GFP expression of RT HEK cells after Dox activation.
The authors acknowledge funding support from the
National Institutes of Health (Grant No. R01EB023776,
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