Animals

Athymic BALB/c nude mice (male, 6–8 weeks old, 20–25 g body weight) were purchased from Vital River (Beijing, China) and housed under pathogen-free conditions. The animal study was performed according to the protocols approved by the Ethics Committee at the General Hospital of the People’s Liberation Army and carried out in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines.

Cell culture, chemical reagents and antibodies

Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were purchased from Cyagen Biosciences (HUXMA-90011, Santa Clara, CA, USA) and were cultured in Dulbecco’s modified Eagle’s Medium (DMEM; Gibco, Grand Island, USA) with low glucose in the presence of 10% fetal bovine serum (FBS; Hyclone, USA). MGC-803 cells were maintained in RPMI 1640 supplemented with 10% FBS. All the cells were incubated in a cell culture incubator at 37 °C in a humidified atmosphere containing 5% CO 2 .

Doxycycline (Dox), puromycin, and G418 were purchased from Sigma Life Science (St. Louis, MO, USA). The rabbit monoclonal antibodies used in this study, including carcinoembryonic antigen (CEA), cytokeratin (CK)7, CK14, CK19, Sonic Hedgehog (Shh), and cyclin D1, and rabbit polyclonal antibody to EDA were purchased from Abcam (Cambridge, MA, USA). The hemagglutinin (HA) mouse monoclonal antibody was supplied by CWBio (Beijing, China).

Single-guide RNA design and lentiviral vector production

Candidate sgRNAs were identified by searching for 5’-N 20 GG motifs, 276 bases to 26 bases upstream of the EDA transcriptional start site (TSS), that conformed with the nucleotide requirements for the spCas9 PAM recognition element (NGG). The sgRNAs were designed using an online CRISPR Design Tool (http://crispr.mit.edu; Fig. 1c).

Fig. 1 Design of CRISPR/dCas9-E nucleases specific for targeting the ectodysplasin (EDA) promotor. a Schematic representation of the EDA genome and b scheme of lentiCRISPR/dCas9-E plasmids. c Schematic representation of the pLKO.1-puro-U6 vector. A guide-sequence insertion site existed downstream of the U6 promoter for cloning the designed single-guide RNA (sgRNA) by the BfuAI restriction site Full size image

The sgRNA expression plasmid (pLKO.1-puro U6 sgRNA BfuAI stuffer) and the plasmid encoding dCas9-E (pHAGE-TRE-dCas9-VP64) were previously developed by others [7] and were obtained from Addgene (plasmids #50920 and #50916). The sgRNAs containing the target sequences were cloned into the pLKO.1-puro-U6 plasmid using the BfuAI sites as described by Kearns et al. [7]. The insertion was verified by clone sequencing.

Lentiviral production

HEK-293FT cells were maintained in DMEM supplemented with 10% FBS. HEK-293FT cells were split and plated in 10 cm2 culture dishes. On the following day, packaging plasmids and dCas9-E or sgRNA-coding plasmids were co-transfected using Lipofectamine® 3000 (Invitrogen) according to the manufacturer’s instructions.

Generation of stable dCas9-E cell lines and co-expression with sgRNAs

The transduction and selection strategy were as previously described [7]. Briefly, BM-MSCs were incubated with tetracycline-responsive element (TRE)-regulated dCas9-E lentivirus on low-attachment plates. The transduced cells were treated with 1 mg/ml G418 from 48 h after transduction to select and maintain stable cell lines. For experiments utilizing sgRNAs, the stable dCas9-E cell lines were infected with sgRNA lentiviruses. Forty-eight hours later, the sgRNA transduced cells were treated with 1 μg/ml puromycin to select sgRNA-expressing cells and 2 μg/ml doxycycline (Dox) to induce expression of dCas9-E (day 0).

Immunofluorescence

Cells were fixed with 4% paraformaldehyde (30 min) and then incubated in 1% bovine serum albumin (BSA) for 1 h. The cells then incubated with primary anti-human antibodies (1:500) overnight at 4 °C. The primary antibodies were decanted and the cells were incubated with phycoerythrin (PE)-conjugated or fluorescein isothiocyanate (FITC)-conjugated goat anti-Rabbit or Mouse IgG secondary antibody (1:1000; Santa Cruz) for 1 h at room temperature in the dark. The cells were finally stained with 4’6-diamidino-2-phenylindole (DAPI; Sigma) and then visualized and examined by a Leica fluorescence microscope.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

RNA was extracted from BM-MSCs and dCas9-E cells using TRIzol reagent (Invitrogen) at 48 h after Dox (2 μg/ml) treatment. Total RNA (1.0 μg) was reverse-transcribed into cDNA using ReverTra Ace qRCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan). Each sample was measured in triplicate. The primer sequences of the genes, including CEA, EDA, CK19, Shh, and cyclin D1, are listed in Additional file 1. All genes were normalized to the endogenous reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative expression levels in cells were calculated as fold changes.

Western blot

BM-MSCs and dCas9-E BM-MSCs were harvested after being treated with Dox (2 μg/ml) for 48 h. Cells were lysed in a passive lysis buffer (Promega, Madison, WI, USA) with a cocktail of protease inhibitors (Roche, Mannheim, Germany) at 4 °C for 30 min and then centrifuged at 12,000 × g for 10 min at 4 °C. Total protein (30 μg) was separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% (w/v) BSA (MP, Auckland, New Zealand) for 1 h, the membranes were probed with primary antibodies overnight at 4 °C. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1.5 h. Specific bands were visualized using a luminol reagent (Santa Cruz Biotechnology).

Transmission electron microscopy (TEM)

After Dox treatment, BM-MSCs and dCas9-E BM-MSCs were harvested and fixed with 2.5% glutaraldehyde in phosphate buffer overnight. Cell samples were first dehydrated by a graded series of ethanol for 15 min at each step and transferred to absolute acetone for 20 min. Resin was then used for sample infiltrating and embedding. Samples were prepared as ultrathin sections and observed with the Hitachi TEM system.

Animal studies

BM-MSCs transfected with dCas9-E and pLKO.1-sgRNAs were implanted into a scald injury animal model. The transplantation procedure was performed as previously described [15]. Briefly, full thickness scald injuries were made on both paws of the hind legs of 10 athymic BALB/c nude mice. The right scalded paw of each mouse received a subcutaneous injection with 1 × 106 Dox-induced dCas9-E BM-MSCs in 100 μl medium. The contralateral (sham) scalded paw was subcutaneously injected with saline. The injury sites were photographed and samples collected every week to measure wound healing and re-epithelialization in each group. Twenty days later an iodine-starch perspiration test was performed, and skin biopsies were also examined by histology. Hematoxylin and eosin staining was used for evaluating the extent of re-epithelialization. Masson and Sirius red staining were used to observe paw fibrosis after injury.

Tumorigenicity tests were generated by subcutaneous injection of either MGC-803 cells (5 × 105 cells) or Dox-induced dCas9-E BM-MSCs (5 × 105 cells) as previously described [19]. Twelve weeks after cell inoculation, hematoxylin and eosin staining was used to measure tumor formation.

Statistical analysis

Statistical analyses were performed using SPSS (V.20) statistical software (SPSS Inc., Chicago, IL, USA). All values are expressed as the mean ± standard deviation. Significant differences were calculated by one-way analysis of variance (ANOVA) followed by the Bonferroni test when performing multiple comparisons between groups. A p value lower than 0.05 was considered as a statistically significant difference.