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Control of adipogenic differentiation in mesenchymal stem cells via endogenous gene activation using CRISPR-Cas9 Yuichi Furuhata, Yuta Nihongaki, Moritoshi Sato, and Keitaro Yoshimoto ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00246 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 29, 2017
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ACS Synthetic Biology
Control of adipogenic differentiation in mesenchymal stem cells via endogenous gene activation using CRISPR-Cas9
1 2 3 4 5 6 7
Yuichi Furuhataa, Yuta Nihongakib, Moritoshi Satob*, Keitaro Yoshimotoc, d*
a
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108-8639, Japan
8 9
b
Department of General Systems Studies, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan
10 11
c
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan
12 13
c
JST, PRESTO, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan
14 15
*
Corresponding authors:
16
Moritoshi Sato, Dr. Sci., Department of General Systems Studies, Graduate School of Arts and
17
Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan
18
Phone and FAX: +81-3-5454-6579
19
E-mail:
[email protected] 20
and
21
Keitaro Yoshimoto, Dr. Sci., Department of Life Sciences, Graduate School of Arts and Sciences,
22
The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan
23
Phone and FAX: +81-3-5454-6580
24
E-mail:
[email protected] 25 26
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Abstract:
2
Mesenchymal stem cells (MSCs) are of interest in regenerative medicine owing to
3
their multilineage differentiation and self-renewal properties. Understanding the in vivo
4
differentiation process is necessary for clinical applications including cell therapy and
5
transplantation. This remains challenging owing to the lack of induction methods that
6
imitate the natural programming process. Endogenous gene regulation of tissue specific
7
transcription factors is therefore desirable. In the present study, we demonstrated
8
endogenous activation of adipogenic genes through the dCas9-based transcription
9
system, and achieved efficient induction of different types of adipocyte-like cells from
10
MSCs. Interestingly, the MSCs converted via single gene activation exhibited
11
morphological and molecular properties of white adipocytes, while beige adipocyte-like
12
cells were induced via multiplex gene activation of three specific transcription factors.
13
These results reveal that the fate of MSCs can be effectively manipulated by direct
14
activation of specific endogenous gene expression using a dCas9-based activator with
15
reduced exogenous additives.
16 17
Keywords:
18
CRISPR-Cas9, Differentiation, Gene activation, Mesenchymal stem cells, White
19
Adipocyte, Beige Adipocyte
20
2
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Introduction
2
Mesenchymal stem cells (MSCs) are one of the most attractive stem cell sources in
3
regenerative medicine; in addition to human induced pluripotent stem (iPS) cells and
4
embryonic stem (ES) cells. While MSCs have multilineage differentiation and
5
self-renewal properties, they are a type of somatic stem cell, and can be obtained from
6
various tissues. These features make them an attractive choice for the development of
7
clinical applications, including cell therapy and transplantation
8
avoid ethical issues and tumorigenesis. Previous studies reported that MSCs can
9
differentiate into various cells, including adipocytes, osteocytes, chondrocytes,
10
myocytes, and neurons 4. To regulate their lineage commitment, several induction
11
methods have been proposed, such as chemically modified medium 5, and ectopic
12
expression of tissue specific transcriptional factors 6. Although these induction methods
13
can efficiently control the fate of MSCs, the associated safety concerns present a
14
formidable obstacle to their therapeutic use. Moreover, since cellular differentiation
15
processes are regulated by complex and dynamic gene expression networks, these
16
methods are not suitable to reproduce the in vivo programming process of MSCs.
17
Therefore, there is a need for a novel method to induce programming of MSCs for
18
therapeutic applications.
19
Clustered
regularly
interspaced
short
palindromic
1–4
, since their use can
repeats
(CRISPR)-Cas9
20
(CRISPR-associated protein 9) technology has been at the forefront in the field of
21
biology due to their potential in biotechnological and medical applications. The
22
catalytically inactivated Cas9 protein derived from Streptococcus pyogenes, called
23
nuclease-dead Cas9 (dCas9), is often used to control gene expression 7. dCas9 forms a
24
complex with single chimeric guide RNA (sgRNAs), and binds to DNA that is 3
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8,9
1
complementary to the first 20 nt of the sgRNA
. Several studies have reported that
2
dCas9 fused with the transcriptional activator domains enables targeted endogenous
3
gene activation
4
and manipulation of dynamic gene programs 7.
8,10,11
. This system has accelerated the study of genome-scale screening
5
In addition to above mentioned applications, dCas9-based transcriptional control also
6
enables cell-fate engineering in the native context. Several groups have demonstrated
7
the direct conversion of primary fibroblasts into myogenic and neurogenic lineages 12,13,
8
and the differentiation control of iPSCs and ES cells
9
expression, dCas9-based endogenous gene activation is suitable for multiple gene
10
activation and can remodel the epigenetic state of the target loci towards differentiation
11
13
12
programming processes, which will allow MSCs to be used in clinical applications.
14–17
. Compared to ectopic
. Therefore, cell-fate engineering via endogenous gene activation imitates native
13
In this study, we first demonstrated dCas9-based transcriptional activation of
14
endogenous genes in MSCs. The expression of adipocyte-specific transcription factors
15
was efficiently activated using a dCas9-based activator. We subsequently attempted
16
cell-fate engineering of MSCs via endogenous gene activation with reduced exogenous
17
additives. We successfully controlled the differentiation of MSCs to white
18
adipocyte-like cells and beige adipocyte-like cells, which are important in disease
19
research for obesity and diabetes.
4
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Results and Discussion
2
Among all published synthetic transcription factors based on dCas9, synergistic
3
activation mediator (SAM) appeared to be the most potent activator 18,19. Therefore, we
4
decided to use SAM for transcriptional activation and cell-fate engineering of MSCs in
5
this study. In the SAM system, dCas9 protein fused with VP64 (dCas9-VP64) form a
6
complex with sgRNA bearing two MS2-binding stemloops (sgRNA 2.0), which recruits
7
MS2 coat protein fused with two distinct transcriptional activators: p65 and heat shock
8
factor 1 (MS2-p65-HSF1).
9
A fluorescent reporter assay was performed to assess and optimize the efficacy of
10
transcriptional activation via SAM in MSCs. We made use of a lentiviral vector carrying
11
the EGFP reporter under the control of a tetracycline-inducible promoter composed of
12
six copies of sgRNA binding sites and a minimal promoter (Figure 1A). Seven days
13
after transduction with the EGFP reporter, MSCs were further lentivirally transduced
14
with dCas9-VP64 and MS2-p65-HSF1 at the same time, followed by selection with
15
blastcidin S, hygromycin B, and puromycin over twenty-one days (Figure 1B and S1).
16
MSCs strongly expressed EGFP four days after sgRNA 2.0 transduction (Figure 1C).
17
Interestingly, despite high multiplicity of infection (MOI), the fluorescence intensity
18
increased with increasing MOI of sgRNA 2.0, suggesting that the amount of
19
intracellular sgRNA 2.0 is important for efficient transcriptional activation in MSCs
20
(Figure S2). SAM mediated gene activation is functional in MSCs, and we therefore
21
decided to perform transduction of sgRNA 2.0 at an MOI of 20 in subsequent studies.
22
For endogenous gene activation in MSCs, MSCs stably expressing dCas9-VP64 and
23
MS2-p65-HSF1, henceforth referred to as SAM MSCs, were established using
24
lentivirus and antibiotic selection. To determine whether SAM MSCs retained 5
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multipotency, osteogenic and adipogenic induction were performed using the relevant
2
induction medium. Alizarin red S staining and Oil red O staining revealed that SAM
3
MSCs grown under osteogenic and adipogenic induction conditions efficiently
4
differentiated into osteocytes and adipocytes, respectively (Figure 2A and B). Moreover,
5
differentiation efficiencies of SAM MSCs were comparable to those of wild-type MSCs.
6
These results indicate that SAM MSCs retain their multipotency.
7
Overexpression of transgenes encoding adipocyte specific transcription factors has 20–22
8
been used to achieve generation of adipocytes from MSCs and fibroblasts
. We
9
hypothesized that the targeted activation of the endogenous genes encoding these
10
factors in their native context could induce adipogenic differentiation of MSCs.
11
Adipogenic lineage commitment of MSCs is mainly regulated by three transcriptional
12
factors; PPARG, CEBPA, and KLF5
13
selected as candidates for the first attempt to induce adipogenesis in MSCs. Since
14
previous studies reported that the level of gene activation by the dCas9-based
15
transcription system can be synergistically enhanced using multiple sgRNAs
16
designed four distinct sgRNAs targeting different positions proximal to the transcription
17
start sites of each gene. Then, we tested whether SAM could activate endogenous genes
18
in MSCs. To activate endogenous gene expression, SAM MSCs were lentivirally
19
transduced with all four sgRNAs for each gene and transduced cells were described as
20
“sgGENE_1-4”. SAM MSCs transduced with sgPPARG_1-4, sgCEBPA_1-4, and
21
sgKLF5_1-4 had 15-, 11110-, and 85-fold upregulation of endogenous gene expression,
22
respectively, compared to cells transduced with control sgRNA (Figure 2C-E).
23
Off-target effect is a general concern to all CRISPR/Cas9-based technology. To validate
24
off-target effects of SAM-mediated gene activation, potential off-target binding sites of
23
. In the present study, these three genes were
10,11
, we
6
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each sgRNA were predicted using CCTop software24. Among the predicted candidates,
2
the site upstream of a transcription start site of a protein coding gene were chosen as the
3
off-target binding site for each sgRNA. The sequences of off-target binding sites were
4
shown in Supporting Information. As shown in Figure S4, we found no strong off-target
5
activation of endogenous genes. These results indicate that a dCas9-based activator can
6
efficiently activate targeted endogenous genes in MSCs.
7
Next, we tested whether endogenous gene activation via SAM can induce adipogenic
8
programming of MSCs. The cells were cultured for 2 weeks in adipocyte maintenance
9
medium after transduction of sgRNAs (Figure 3A). To assess adipogenic phenotypes,
10
the production of oil droplets in the treated MSCs was analyzed by Oil red O staining,
11
which is used to detect triacylglycerol accumulation. Though there were no oil droplets
12
observed in mock-, sgControl-, and sgKLF5_1-4-transduced cells (Figure 3B), a
13
number
14
sgCEBPA_1-4-transduced cells (Figure 3C). Surprisingly, these gene-activated cells
15
produced as many droplets as adipocytes induced using adipogenic induction medium
16
(Figure 3B and C). To determine the efficiency of adipogenic differentiation, Oil red O
17
staining intensities were quantified by extracting Oil red O using isopropyl alcohol.
18
Both sgPPARG_1-4- and sgCEBPA_1-4-transduced cells had high Oil red O intensities,
19
similar to those of cells cultured in adipogenic induction medium (Figure 3D). Both
20
sgPPARG_1-4- and sgCEBPA_1-4-transduced cells produced the same number of oil
21
droplets as cells cultured in adipogenic induction medium (Figure 3E).
of
droplets
were
observed
in
both
sgPPARG_1-4-
and
22
For detailed characterization of induced adipocytes, we focused on a panel of
23
adipocyte-associated genes. Real-time RT PCR was carried out to measure the
24
expression levels of adipogenic marker genes, including PPARG, CEBPA, ADIPOQ, 7
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FABP4, FASN, and LIPE. Adipocyte marker genes were not expressed in the
2
undifferentiated samples (mock- and sgControl-transduced cells). In contrast, both
3
sgPPARG_1-4- and sgCEBPA_1-4-transduced cells had much higher levels of
4
expression than undifferentiated cells at day 14, which was comparable with that of
5
cells cultured in adipogenic induction medium. These findings suggest that dCas9-based
6
transcriptional activation can induce adipogenic differentiation of MSCs, even in
7
adipocyte maintenance medium. Adipocyte maintenance medium does not contain
8
indomethacin and 3-isobutyl-1-methylxanthine, which act as a PPARγ agonist and an
9
enhancing agent for dexamethasone, respectively. Therefore, we accomplished efficient
10
adipogenic differentiation of MSCs using endogenous gene activation with reduced
11
exogenous additives.
12
Mammalian adipose tissue is divided into white, beige, or brown. White adipocytes,
13
which are specialized for energy storage and cause metabolic disease including obesity
14
and diabetes, have a single, large deposit of lipids. In contrast, beige and brown
15
adipocytes have multiple, small oil droplets in the cytosol, and can counteract metabolic
16
disease through thermogenesis 25. In the human body, the metabolic profile is regulated
17
by the balance of cellular activities in these three adipocytes. Understanding how these
18
adipocytes are induced and characterized in the natural programming process is helpful
19
for the study of disease mechanisms, and could contribute to a cell-based drug screening
20
for metabolic diseases. Ahfeldt et al. reported that overexpression of PPARG transgene
21
alone or combined with CEBPB and PRDM16 transgenes in MSCs derived from iPS
22
cells induced white or brown adipogenesis, respectively 21. On the other hand, the other
23
studies showed that brown adipocytes were derived from skeletal muscle precursor cells
24
rather than white adipocyte precursor cells, while beige adipocytes were come from 8
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white adipocyte precursor cells26,27. In the present study, we tested whether SAM can
2
activate at multiple endogenous loci in MSCs and regulate their lineage commitment
3
toward beige or brown adipocytes. Four distinct sgRNAs, each targeting the promoter
4
region of CEBPB and PRDM16, were designed. In a single-gene activation experiment,
5
the endogenous gene activation of each gene was observed (Figure 4A). Next, multiple
6
activation of PPARG, CEBPB, and PRDM16 genes was carried out in SAM MSCs. In
7
the sample transduced with multiple sgRNAs targeting these three genes, we observed
8
successful activation of all target genes, although all genes exhibited a small decrease in
9
the magnitude of upregulation compared to that observed in single gene activation
10
(Figure 4B).
11
To control the adipogenic differentiation among white and beige or brown adipocytes,
12
SAM MSCs were transduced with sgRNAs targeting these three genes independently
13
and together. After culturing in adipocyte maintenance medium for three weeks, cells
14
were stained with Nile red. The cells transduced with multiple sgRNAs targeting three
15
genes (sgMixture) had characteristic beige or brown adipocyte morphology, with a
16
number of small droplets (Figure 4C and D). In contrast, the cells transduced with only
17
sgPPARG_1-4 formed a single large droplet, similar to white adipocytes. Interestingly,
18
there were some beige or brown adipocyte-like cells in SAM MSCs transduced with
19
sgRNAs targeting only PRDM16 (Figure 4C). For characterization of induced
20
adipocytes, real-time RT PCR was carried out to measure the expression levels of beige
21
selective marker genes; TMEM26 and SHOX2, and brown selective marker genes;
22
OPLAH and SLC29A1. As shown in Figure 4E, the gene expression of beige selective
23
markers
24
sgPPARG_1-4-transduced cells. In contrast, that of brown selective markers was not
was
upregulated
in
sgMixture-transduced
cells
compared
to
9
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upregulated. These findings suggest that induced adipocytes with a number of small
2
droplets in this study are beige adipocytes rather than brown adipocytes. We have
3
therefore confirmed that the dCas9-based transcription system can simultaneously
4
activate multiple genes in MSCs. Furthermore, we found that it is possible to precisely
5
regulate the cell fate of MSCs between white and beige adipocyte-like cells, through
6
endogenous gene control.
7
In the present study, we established that adipogenic programming of MSCs can be
8
controlled through targeted activation of endogenous genes. A highly efficient
9
CRISPR-Cas9-based transcription activator, named SAM, was introduced into MSCs
10
via lentiviral vectors, and demonstrated robust activation of endogenous adipogenic
11
factors. Individual endogenous activation of both PPARG and CEBPA efficiently
12
induced white adipocyte-like cells from MSCs, which exhibited morphological and
13
molecular properties of adipocytes. Beige adipocyte-like cells were also induced via
14
multiplex activation of endogenous PPARG, CEBPB, and PRDM16, and differentiation
15
control between white and beige adipocyte-like cells was achieved by changing only the
16
sgRNA target sequences. The balance of cellular activities between white and beige
17
adipocytes is closely related to metabolic diseases such as obesity and diabetes. It is still
18
challenging to induce individual homogenous cell populations of white and beige
19
adipocytes using chemically modified medium. Therefore, the cells induced in the
20
present study may provide opportunities for the development of new therapeutic
21
strategies for managing these diseases.
22
In cell fate engineering using ectopic expression of transcriptional factors, the genes
23
targeted are often limited by their large size, which causes difficulty in cDNA cloning
24
and transfection into cells. Since an sgRNA expressing cassette is small and easily 10
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introduced into cells, any gene can be activated using dCas9-based transcriptional
2
factors. The sgRNA dependent transcriptional activation also provides temporal
3
regulation of targeted gene expression by control of sgRNA introduction time.
4
Furthermore, the greater part of endogenous gene has several isoforms and the
5
coordinated expressions of them are important for regulating diverse biological
6
phenomena including differentiation. dCas9-based endogenous gene activation enables
7
the induction of all promoter-based products in a similar way as a native transcriptional
8
process, whereas ectopic overexpression allows the induction of only one isoform of
9
interest. These features of dCas9-based transcriptional activation may help to reproduce
10
the complex gene regulatory networks in natural programming processes.
11
Lineage reprogramming has emerged as a promising approach for generating desired
12
cell types and how to obtain desirable cell types is one of the important questions in
13
regenerative medicine. A lot of works have shown that the ectopic expression of
14
multiple transcription factors promotes the differentiation of stem cells into multiple cell
15
types. However, there are difficulties in manipulating multiple gene expression and
16
identifying an optimal combination of transcription factors due to the complexity of the
17
cDNA cloning and ectopic gene introduction. In contrast, dCas9-based endogenous
18
gene activation is suitable for multiple gene activation. The SAM MSC platform shown
19
in this study should be useful for identification of an optimal combination of
20
transcription factors to convert MSCs to various cells such as osteocytes, chondrocytes,
21
myocytes, neurons, and other cells.
22 23 24
Conclusion In conclusion, we demonstrate dCas9-based transcriptional activation in MSCs, 11
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which enabled cell-fate engineering of MSCs with reduced exogenous additives. Since
2
endogenous gene activation can induce their differentiation in the native context, this
3
approach improves the usefulness of MSCs as a cell source for clinical applications.
4
Furthermore, it has wide applicability for modulating the cell fate of MSCs into various
5
lineages, and may be useful to determine how MSCs develop into several lineages in
6
vivo.
7 8 9
Methods
10
Cell Culture
11
Cells were cultured at 37 °C under 5% CO2 in all experiment. UE7T-13 (JCRB) cells
12
were used as MSCs in the present study, and were cultured in Dulbecco's modified
13
Eagle medium (Wako Pure Chemical Industries) supplemented with 10% (v/v) fetal
14
bovine serum (FBS, Biosera) and 1% (v/v) Penicillin-Streptomycin-Neomycin (PSN)
15
antibiotic mixture (Thermo Fisher Scientific). HEK293T/17 cells (ATCC) and Lenti-X™
16
293T cells (TAKARA BIO) were cultured in Dulbecco’s Modified Eagle Medium
17
(DMEM,
18
Penicillin-Streptomycin (Thermo Fisher Scientific Inc.).
Sigma-Aldrich)
supplemented
with
10%
FBS
(Hyclone),
1%
19 20
Establishment of stable cell lines
21
To establish stably expressing MSC cell-lines, UE7T-13 cells were plated at
22
approximately 1.0 × 105 cells/dish in 100 mm dishes, and were transduced with the
23
indicated lentiviral vectors with 0.8 µg/ml hexadimethrine bromide. Forty-eight hours
24
after transduction, the culture medium was changed and antibiotics were added. The 12
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subsequent duration of antibiotic selection was at least 2 weeks. For antibiotic selection
2
in UE7T-13 cells, antibiotics were used at the following concentrations: blasticidin S
3
(Wako Pure Chemical Industries) 10 µg/ml, hygromycin B (Wako Pure Chemical
4
Industries) 1000 µg/ml, and puromycin 0.5 µg/ml (Wako Pure Chemical Industries).
5 6
Reporter gene activation
7
EGFP reporter SAM MSCs were plated at approximately 2.0 × 103 cells/well in
8
96-well plates and cultured for 24 h. The cells were then transduced with lentiviral
9
vector encoding the indicated sgRNA at the indicated multiplicity of infection (MOI)
10
with 0.8 µg/ml hexadimethrine bromide. The culture medium was changed 48 h after
11
transduction. After incubation for 48 h, samples were washed twice with PBS, stained
12
with 10 µg/ml Hoechst 33258 (Promocell), and observed using a fluorescent microscope
13
(Olympus). For quantification, fluorescent intensities of EGFP were measured using
14
ImageJ, normalized to Hoechst 33258, and then standardized to that of the
15
mock-infected samples.
16 17
Endogenous gene activation
18
SAM MSCs were plated at approximately 2.0 × 103 cells/well in 96-well plates and
19
cultured for 24 h. The cells were then transduced with lentiviral vector encoding the
20
indicated sgRNA with 0.8 µg/ml hexadimethrine bromide. The total MOI was 20. For
21
simultaneous activation of three genes, the ratio of sgRNAs targeting each gene was
22
1:1:1. The culture medium was changed 48 h after transduction. After incubation for 48
23
h, total RNA samples were extracted for quantitative PCR analysis using RNeasy Mini
24
Kit (Qiagen). 13
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1 2
Real-time reverse transcription PCR
3
Reverse transcription reaction was performed using the ReverTra Ace qPCR RT
4
Master Mix with gDNA Remover (Toyobo). Real-time RT PCR was performed using
5
THUNDERBIRD SYBR qPCR Mix (Toyobo) with StepOnePlusTM (Thermo Fisher
6
Scientific). Additional experimental procedures can be found in the Supplemental
7
Experimental Procedures.
8 9
Adipogenic induction using gene activation
10
SAM MSCs were plated at approximately 2.0 × 103 cells/well in 96-well plates and
11
cultured for 24 h at 37 °C in 5% CO2. sgRNA transduction was performed in the same
12
way as Endogenous gene activation. Forty-eight hours after transduction, the medium
13
was changed to adipocyte maintenance medium (DMEM supplemented with 10% (v/v)
14
FBS, 1% (v/v) PSN, 1 µM dexamethasone (Wako Pure Chemical Industries) and 10
15
µg/ml insulin (Wako Pure Chemical Industries)) for two or three weeks for white or
16
beige adipogenesis, respectively. The medium was changed every four or seven days for
17
white adipogenesis or beige adipogenesis, respectively.
18 19
Cell staining
20
For an osteogenic differentiation assay, 4% PFA fixed cells were stained with Alizarin
21
red S (Wako Pure Chemical Industries). For adipogenic differentiation assays, 4% PFA
22
fixed cells were stained with Oil red O (Wako Pure Chemical Industries) or Nile Red
23
(Wako Pure Chemical Industries). Additional experimental procedures can be found in
24
the Supplemental Experimental Procedures. 14
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Quantification and Statistical Analysis
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All measurements are presented as mean ± standard error (SE). Statistical
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significance was evaluated using independent samples Student’s t-test. Differences
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between groups were defined as statistically significant when *p < 0.05, or **p < 0.01.
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Sample sizes are indicated in the Figure legends. Statistical details are described in
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Method Details and Figure legends.
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Author contributions
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Y. F., Y. N., M. S., and K. Y. conceived the project. Y. F., Y. N., M. S., and K. Y
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designed experiments. Y. F. and Y. N. performed experiments. Y. F. analyzed data. Y. F.
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and K. Y. wrote the manuscript. All authors edited the manuscript.
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Acknowledgements
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This work was partially supported by the JSPS KAKENHI [grant numbers 15K13721],
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JST-PRESTO [grant number JPMJPR16FB], JST-CREST [grant number JPMJCR1653],
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JST-START, and JSPS Research Fellowships for Young Scientists [grant number
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15J08446 and 15J05897].
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Conflict of Interest
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The authors declare no competing financial interest.
16 17 18
Abbreviations
19
Mesenchymal stem cells (MSCs), Clustered regularly interspaced short palindromic
20
repeats (CRISPR)-Cas9 (CRISPR-associated protein 9), nuclease-dead Cas9 (dCas9),
21
single chimeric guide RNA (sgRNAs), synergistic activation mediator (SAM), dCas9
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protein fused with VP64 (dCas9-VP64), sgRNA bearing two MS2-binding stemloops
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(sgRNA 2.0), MS2 coat protein fused with two distinct transcriptional activators: p65
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and heat shock factor 1 (MS2-p65-HSF1), MOI (multiplicity of infection), proliferator 16
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activated receptor gamma (PPARG), CCAAT/enhancer binding protein alpha (CEBPA),
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kruppel like factor 5 (KLF5), adiponectin (ADIPOQ), fatty acid binding protein 4
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(FABP4), fatty acid synthase (FASN), lipase E, hormone sensitive type (LIPE),
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CCAAT/enhancer binding protein beta (CEBPB), PR/SET domain 16 (PRDM16),
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transmembrane protein 26 (TMEM26), short stature homeobox 2 (SHOX2),
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5-oxoprolinase (ATP-hydrolysing) (OPLAH), solute carrier family 29 member 1
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(augustine blood group) (SLC29A1), and glyceraldehyde-3-phosphate dehydrogenase
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(GAPDH)
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Supporting Information
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Supporting Information includes supporting methods, four figures and four tables and
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can be found with this article online at the publisher’s website.
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Supporting Methods
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Figure S1. The lentiviral constructs utilized in this study. Related to Figures 1-4.
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Figure S2. The relationship between fluorescence intensity and MOI of sgRNA 2.0
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lentivirus. Related to Figure 1.
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Figure S3. Maps showing the sgRNA targeting sites. Related to Figures 1-4.
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Figure S4. Off-target analysis. Related to Figures 2-4.
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Table S1. Target sequences of sgRNAs. Related to Figures 1-4.
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Table S2. Primer sequences used in the analysis of mRNA expression by real-time RT
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PCR. Related to Figures 2-4.
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Table S3. Medium compositions used in this study. Related to Figures 2-4.
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Table S4. The sequences of off-target binding sites. Related to Figures 2-4. 17
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Supporting References
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References
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Figure Legends
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Fig. 1 Reporter Gene Activation by CRISPR-Cas9-based Transcription System in
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MSCs.
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(A) Schematic diagram of the EGFP reporter. (B) Schematic diagram of the
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experimental
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CRISPR-Cas9-based transcription system in MSCs. (C) Exogenous EGFP activation in
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MSCs. MSCs were transduced with indicated sgRNA 2.0 lentivirus at an MOI of 20.
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Four days post transduction, samples were stained with Hoechst 33258. Scale bar
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procedure
used
for
reporter
gene
activation
assay
by
a
represents 100 µm. See also Figure S2.
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Fig. 2 Multipotency and Endogenous Gene Activation of SAM MSCs.
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(A) Alizarin red S staining of osteogenic-induced MSCs. Wild-type and SAM MSCs
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were cultured using the osteogenic induction medium. Scale bar represents 50 µm. (B)
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Oil red O staining of adipogenic-induced MSCs. Wild-type and SAM MSCs were
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cultured using the adipogenic induction medium. Scale bar represents 50 µm. (C-E)
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Endogenous gene activation of (C) PPARG, (D) CEBPA, and (E) KLF5 in SAM MSCs
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with targeted activation. SAM MSCs were transduced with indicated sgRNA 2.0
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lentivirus at an MOI of 20. mRNA levels were measured four days post transduction.
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The mRNA expression levels were normalized by GAPDH and then standardized to that
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in the sample of sgControl. Values shown are the mean ± SE of n = 3. *p < 0.05, **p