Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmdmdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system.
Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmdmdx mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.
Duchenne muscular dystrophy (DMD) is one of the most common muscular dystrophies and is characterized by the absence of dystrophin, affecting 1 of approximately 5,000 newborn boys worldwide1. Loss of dystrophin gene function results in structural muscle defects leading to progressive myofibers degeneration1,2. Recombinant adeno-associated virus (rAAV)-mediated gene therapy system has been tested to restore the dystrophin expression and improve muscle function, such as gene replacement using micro-dystrophins (µ-Dys). However, the rAAV approach requires repeated injections to sustain expression of the functional protein3,4. Therefore, we need a strategy that can provide effectively and permanently recover dystrophin gene expression in patients with DMD. The Dmdmdx mouse, a mouse model for DMD, has a point mutation in exon 23 of the dystrophin gene that introduces a premature termination codon and results in a non-functional truncated protein lacking the C-terminal dystroglycan binding domain. Recent studies demonstrated the use of CRISPR/Cas9 technology to restore dystrophin gene expression by accurate gene correction or mutant exon deletion in small and large animal5,6,7. Long et al.8 reported the method for correcting the dystrophin gene mutation in Dmdmdx mouse germline by homology-directed repair (HDR) based CRISPR/Cas9 genome editing. El Refaey et al.9 reported that rAAV could efficiently excise the mutant exon 23 in dystrophic mice. In these studies, gRNAs were designed in the introns 20 and 23 to cause double-stranded DNA breaks, which partially restored the dystrophin expression after DNA repair via non-homologous end joining (NHEJ). Even more exciting, Amoasii et al.10 recently reported the efficacy and feasibility of rAAV-mediated CRISPR gene editing in restoring dystrophin expression in canine models, an essential step in future clinical application.
DMD also causes stem cell disorders11. For muscle damage, residential muscle stem cells replenish dying muscle cells after muscle differentiation. However, the consecutive cycles of injury and repair lead to shortening of telomeres in muscle stem cells12, and premature depletion of stem cell pools13,14. Therefore, a combination of autologous stem cell therapy with genome editing to restore dystrophin expression can be a practical approach for treating DMD. The CRISPR/Cas9 technology provides the possibility of generating autologous genetically corrected induced pluripotent stem cells (iPSC) for functional muscle regeneration and prevent further complications of DMD without causing immune rejection. However, iPSCs have a risk of tumor formation, which could be alleviated by the differentiation of iPSC into myogenic progenitor cells.
In this protocol, we describe the use of non-integrating Sendai virus to reprogramming dermal fibroblasts of Dmdmdx mice into iPSCs and then recover dystrophin expression by CRISPR/Cas9 genome deletion. After validation of Exon23 deletion in iPSC by genotyping, we differentiated genome-corrected iPSC into myogenic progenitors (MPC) via MyoD-induced myogenic differentiation.
All animal handling and surgical procedures were performed by a protocol approved by the Augusta University Institutional Animal Care and Use Committee (IACUC). Mice were fed standard diet and water ad libitum.
1. Isolation of primary mouse fibroblasts from adult Dmdmdx mice
2. Reprogramming mouse skin fibroblasts into iPSCs
3. Using alkaline phosphatase live stain and flow cytometry to quantify reprogramming efficiency
4. Selecting and harvesting ES-like cells
5. Freezing iPSCs for cryopreservation
6. Immunofluorescence staining for stem cell markers in iPSCs
7. Investigating the pluripotency of iPSCs in vivo
8. Construction of CRISPR/Cas9 lentiviral vector targeting introns flanking dystrophin exon 23
9. Lentiviral vector packaging
10. Concentration and purification of lentiviral vectors
11. Deletion of exon 23 in mouse iPSCs with two guide RNAs (gRNAs) coupled with Cas9
12. Identification of iPSC colonies with exon23 deletion
13. Using the Tet-on MyoD activation system to directly differentiate iPSC into myogenic progenitor cells (MPC)
14. Quantitative reverse transcription PCR for evaluating dynamic muscle differentiation and DMD exon 22-24 expression
15. Immunofluorescence staining of myosin heavy chain 2 (MYH2) and dystrophin protein expression
Establishment of Dmdmdx skin fibroblasts derived iPSC. We demonstrated the efficiency of generating mouse iPSCs from Dmdmdx mice derived skin fibroblast using the integration-free reprogramming vectors. Figure 1A demonstrated that the appearance of embryonic stem cell (ESC)-like colonies at three weeks after infection. We evaluate the efficiency of iPSC induction by live alkaline phosphatase (AP) stain; Figure 1B shows that the percentage of AP-positive cells was around 1.8% by FACS analysis. SSEA1, Lin28, Nanog, OCT4 and SOX2, pluripotency markers for mouse embryonic stem cells, were positive for iPSC colonies by immunofluorescent staining, (Figure 1C). To investigate the three germline differentiation of iPSCs in vivo, we intramuscularly injected iPSCs into the mouse gastrocnemii. We observed that the injected iPSCs differentiated into liver cells (endoderm), smooth muscle cells (mesoderm), and adrenergic neuron cells (ectoderm) (Figure 1D), indicting the pluripotency of iPSCs.
CRISPR/Cas9-mediated exon23 deletion. We designed two guide RNAs that flank the mutant exon 23. After Cas9-mediated double-stranded breaks (DSB) and non-homologous end joining (NHEJ), mutant exon 23 was deleted, allowing for truncated but functional dystrophin production (Figure 2A). To identify exon 23 deleted mouse iPSC, cells were sparsely seeded, and individual colonies were picked and propagated. Genomic DNAs extracted from these colonies were subjected to PCR genotyping. Figure 2B demonstrated that colony #1 and #2 have exon 23 deletions indicating a successful deletion of the exon 23.
Differentiating mouse iPSCs into a myogenic lineage and restoring dystrophin expression. We use a tetracycline-inducible MyoD expression system to induce myogenic differentiation of iPSCs. Doxycycline was used to induce MyoD expression in iPSCs. Figure 3A shows the time course of muscle differentiation in Dox-treated iPSCs. qRT-PCR showed that the mRNA level of OCT4, a pluripotent marker, gradually decreased, while the expression of ACTA1, a skeletal muscle marker, increased after Dox induction. Also, we observed the myotubes formation at two weeks after Dox treatment (Figure 3B). Importantly, the qRT-PCR assay showed the recovery of DMD exon 24 mRNA expression in Dox-induced, Cas9-mediated Exon23 deleted line in comparison to Cas9-control line (Figure 3C). Inconsistent with qRT-PCR, immunofluorescent staining shows the dystrophin protein expressionin Cas9-mediated exon 23 deleted cells, whereas the dystrophin expression was absent in control cells (Figure 3D).
Figure 1: Reprogramming skin fibroblasts from Dmdmdx mice into iPSCs.
(A) Representative image of ES-like colonies (scale bar = 200 µm). (B) FACS analysis of the reprogramming efficiency of mouse skin fibroblasts into iPSCs after 8 days of Sendai virus transduction by live AP staining. (C) Immunofluorescent staining of SSEA1, Lin28, Nanog, Oct4, and SOX2 in iPSCs (scale bar = 50 µm). (D) Immunofluorescent staining for AFP (endoderm), SMA (mesoderm), and tyrosine hydrolase (TH) (ectoderm) of teratoma 2 weeks after iPSC injection into gastrocnemii (scale bar = 20 µm). Please click here to view a larger version of this figure.
Figure 2: CRISPR/Cas9-mediated exon23 deletion.
(A) Schematic diagram of CRISPR/Cas9-mediated exon 23 deletions. The Cas9 nuclease targets intron 22 and intron 23 by two gRNAs. Double-stranded breaks (DSBs) by Cas9 results in the excision of the mutant exon 23. The distal ends are repaired by non-homologous end joining (NHEJ), resulting in the restoration of the reading frame of the dystrophin gene. (B) PCR genotyping analysis of exon 23. The arrow indicates the PCR product of exon 23. GAPDH serves as a reference. Please click here to view a larger version of this figure.
Figure 3: Differentiating mouse iPSCs into the myogenic lineage and restoring dystrophin expression.
(A) qRT-PCR showed the time course of mRNA level of Oct4 and ACTA1 in Dox-treated exon 23-deleted Dmdmdx iPSC (*P < 0.05 vs D0, D6, D10, #P < 0.05 vs D0, D3, D10, $P < 0.05 vs D0, D3, D6, n = 4 for Oct4) (*P < 0.05 vs D6 and D10, #P < 0.05 vs D0, D3, and D10, $P < 0.05 vs D0, D3, and D6, n = 3 for ACTA1). (B) Left: Representative image of myotube formation from Dox-induced mouse iPSCs (scale bar = 200 µm). Right: Immunofluorescent analysis of MYH2 in myotube formation from Dox-induced mouse iPSCs (scale bar = 20 µm). (C) Upper: the PCR primer positions for DMD Exon22, Exon23 and Exon24; Bottom: qRT-PCR analysis of the mRNA level of DMD Exon22, Exon23, and Exon24 expression in MPC (****P < 0.0001, n = 3). (D) Immunofluorescent analysis of dystrophin expression in Dox-induced MPC from iPSCCas9-Ctrl and iPSCCas9-gRNA (scale bar = 50 µm). Please click here to view a larger version of this figure.
Guide primers | |
i22 sense | 5'-CACCGTTAAGCTTAGGTAAAATCAA- 3' |
i22 antisense | 5’-AAACTTGATTTTACCTAAGCTTAAC-3’ |
i23 sense | 5'-CACCGAGTAATGTGTCATACCTTCT- 3' |
I23 antisense | 5’-AAACAGAAGGTATGACACATTACTC-3’ |
PCR primers | |
OCT4-Forward | 5'-AGCTGCTGAAGCAGAAGAGGATCA-3' |
OCT4-Reverse | 5'-TCTCATTGTTGTCGGCTTCCTCCA-3' |
ACTA1-Forward | 5'-GATCCATGAGACCACCTACAAC-3' |
ACTA1-Reverse | 5'-TCAGCGATACCAGGGTACAT-3' |
Exon22-Forward | 5'-TTACCACCAATGCGCTATCA-3' |
Exon22-Reverse | 5'-CCGAGTCTCTCCTCCATTATTTC-3' |
Exon23-Forward | 5'-CCAAGAAAGCACCTTCAGAAATATG-3' |
Exon23-Reverse | 5'-TTTGGCAGCTTTCCACCA-3' |
Exon24-Forward | 5'-AAC CTT ACA GAA ATG GAT GGC-3' |
Exon24-Reverse | 5'-TTTCAGGATTTCAGCATCCC-3' |
GAPDH-Forward | 5'-TGACAAGCTTCCCATTCTCG-3' |
GAPDH-Reverse | 5'-CCCTTCATTGACCTCAACTACAT-3' |
Table 1: Primer sequence.
Duchenne Muscular Dystrophy (DMD) is a destructive and ultimately fatal hereditary disease characterized by a lack of dystrophin, leading to progressive muscle atrophy1,2. Our results demonstrate the restored dystrophin gene expression in Dmdmdx iPSC-derived myogenic progenitor cells by the approach of CRISPR/Cas9-mediated exon23 deletion. This approach has three advantages.
First, we generated iPSCs from Dmdmdx mouse-derived dermal fibroblasts using a non-integrated RNA vector. A variety of methods have been developed to generate iPSCs, such as lentiviral and retroviral vectors, which will integrate into host chromosomes to express reprogramming genes, thus bearing safety concerns. DNA-based vectors such as plasmid vectors, adeno-associated viruses and adenoviruses exist in a non-integrated manner; however, they may still integrate into the host chromosome at a low frequency. In this study, we used a modified, non-transmissible Sendai virus, a non-integrated RNA vector, to safely and effectively deliver stem cell transcription factors to fibroblasts for reprogramming.
Next, we use CRISPR-mediated genome deletion, rather than CRISPR/Cas9-mediated precision gene correction, to restore dystrophin expression in iPSCs. This method is feasible and efficient; it is easy to design multiple gRNAs to delete multiple mutant exons, which occur in many human DMD patients17. Exon deletion utilizes a relatively efficient non-homologous end joining pathway, and the method also avoids the need to deliver a DNA repair template. Therefore, in comparison to Cas9-mediated precision correction, Cas9–mediated exon deletion is suitable for DMD patients with multiple gene mutations.
Finally, we induced undifferentiated iPSCs into myogenic progenitor cells, which may reduce the risk of tumorigenesis caused by iPSCs. In this protocol, we induced MyoD expression via an inducible tetracycline-regulated (Tet-On) vector system to differentiate iPSCs into skeletal muscle progenitors18,19.
In conclusion, the combination of CRISPR/Cas9 genome editing with Tet-on MyoD activation system may provide a safe, feasible, and efficient strategy for mutant DMD-Exon23 deletion in stem cells for cell transplantation in DMD patients.
To select and harvest ES-like cells efficiently, we should identify the undifferentiated iPSC cells via their dome-like morphology, and an inking object marker can help us label individual clones from the bottom of the culture dish with a 1.8 mm circle around the iPSC clones. To avoid leakage of trypsin solution, we need to apply grease evenly to the bottom of rings. Also, after placing the grease-coated rings on the top of the labeled cell colonies, care should be taken not to touch the rings. Otherwise, the iPSC clones will be detached.
The protocol has its limitations; for example, we chose a non-integrated RNA vector system to generate iPSCs. However, we used a lentiviral CRISPR/Cas9 system to delete DMD exon 23 and a lentiviral-based MyoD activation system to induce iPSC myogenic differentiation; these integrative lentiviral vectors have safety concerns. However, these issues can be solved by the application of a ribonucleoprotein (RNP) complex comprising a recombinant, high-purity S. pyogenes Cas9 nuclease with a crRNA:tracrRNA duplex; we can choose chemically modified MyoD mRNA transfection to directly differentiate iPSCs into myogenic progenitors, although the efficiency may be challenging.
The authors have nothing to disclose.
Tang and Weintraub were partially supported by NIH-AR070029, NIH-HL086555, NIH-HL134354.
Surgical Instruments | |||
31-gauge needle | Various | ||
Sharp Incision | Various | ||
Sterile Scalpels | Various | ||
Tweezers | Various | ||
Fibroblast medium (for 100 mL of complete medium) | Company | Catalog Number | Volume |
2-Mercaptoethanol (55 mM) | Gibco | 21-985-023 | 0.1 mL |
Antibiotic Antimycotic Slution 100x | CORNING | MT30004CI | 1 mL |
Dulbecco's Modified Eagle's Medium – high glucose | SIGMA | D6429 | 87 mL |
Fetal Bovine Serum Characterized | HyClone | SH30396.03 | 10 mL |
L-Glutamine solution | SIGMA | G7513 | 1 mL |
MEM Non-Essential Amino Acids Solution (100x) | Gibco | 11140076 | 1 mL |
TVP solution (for 500 mL of complete solution) | Company | Catalog Number | Volume |
Chicken Serum | Gibco | 16110-082 | 5 mL |
EDTA | Sigma-Aldrich | E6758 | 186 mg |
Phosphat-buffered saline | to 500 mL | ||
Trypsin (2.5%) | Thermo | 15090046 | 5 mL |
mES growth medium(for 500 mL of complete solution) | Company | Catalog Number | Volume |
2-Mercaptoethanol (55 mM) | Gibco | 21-985-023 | 0.5 mL |
Antibiotic Antimycotic Slution 100x | CORNING | MT30004CI | 5 mL |
Dulbecco's Modified Eagle's Medium – high glucose | SIGMA | D6429 | 408.5 mL |
Fetal Bovine Serum Characterized | HyClone | SH30396.03 | 75 mL |
L-Glutamine solution | SIGMA | G7513 | 5 mL |
Mouse recombinant Leukemia Inhibitory Factor (LIF), 0.5 x 106 U/mL | EMD Millipore Corp | CS210511 | 500 μL |
MEK/GS3 Inhibitor Supplement | EMD Millipore Corp | CS210510-500UL | 500 μL |
MEM Non-Essential Amino Acids Solution (100x) | Gibco | 11140076 | 5 mL |
The ES cell media should not be stored for more than 4 weeks and with inhibitors not more than 2 weeks. | |||
mES frozen medium(for 50 mL of complete solution) | Company | Catalog Number | Volume |
Dimethyl sulfoxide (DMSO) | SIGMA | D2650 | 5 mL |
Dulbecco's Modified Eagle's Medium – high glucose | SIGMA | D6429 | 24.9 mL |
Fetal Bovine Serum Characterized | HyClone | SH30396.03 | 25 mL |
Mouse recombinant Leukemia Inhibitory Factor (LIF), 0.5 x 106 U/mL | EMD Millipore Corp | CS210511 | 50 μL |
Name of Material/ Equipment | Company | Catalog Number | RRID |
0.05% Trypsin/0.53 mM EDTA | CORNING | 25-052-CI | |
4% Paraformaldehyde | Thermo scientific | J19943-k2 | |
Accutase solution | SIGMA | A6964 | Cell detachment solution |
AgeI-HF | NEB | R3552L | |
Alexa488-conjugated goat-anti-mouse antibody | Invitrogen | A32723 | AB_2633275 |
Alexa488-conjugated goat-anti-rabbit antibody | Invitrogen | A32731 | AB_2633280 |
Alexa555-conjugated goat-anti-rabbit antibody | Invitrogen | A32732 | AB_2633281 |
anti-AFP | Thermo scientific | RB-365-A1 | AB_59574 |
anti-α-Smooth Muscle Actin (D4K9N) XP | CST | 19245S | AB_2734735 |
anti-Dystrophin | Thermo | PA5-32388 | AB_2549858 |
anti-LIN28A (D1A1A) XP | CST | 8641S | AB_10997528 |
anti-MYH2 | DSHB | mAb2F7 | AB_1157865 |
anti-Nanog-XP | CST | 8822S | AB_11217637 |
anti-Oct-4A (D6C8T) | CST | 83932S | AB_2721046 |
anti-Sox2 | abcam | ab97959 | AB_2341193 |
anti-SSEA1(MC480) | CST | 4744s | AB_1264258 |
anti-TH (H-196) | SANTA CRUZ | sc-14007 | AB_671397 |
Alkaline Phosphatase Live Stain (500x) | Thermo | A14353 | |
Blasticidin S | Sigma-Aldrich | 203350 | |
BsmBI/Esp3I | NEB | R0580L/R0734L | |
Carbenicillin | Millipore | 205805-250MG | |
Collagenase IV | Worthington Biochemical Corporation | LS004189 | |
Competent Cells | TakaRa | 636763 | |
CutSmart | NEB | B7204S | |
CytoTune-iPS 2.0 Sendai Reprogramming Kit | Thermo | A16517 | |
DirectPCR Lysis Reagent (cell) | VIAGEN BIOTECH | 302-C | |
Dispase (1 U/mL) | STEMCELL Technologies | 7923 | |
Doxycycline Hydrochloride | Fisher BioReagents | BP26535 | |
EcoRI-HF | NEB | R3101L | |
Fibronectin bovine plasma | SIGMA | F1141 | |
QIAEX II Gel Extraction Kit (500) |
QIAGEN | 20051 | |
Gelatin from porcine skin, type A | SIGMA | G1890 | |
HardSet Antifade Mounting Medium with DAPI | Vector | H-1500 | |
Hygromycin B (50 mg/mL) | Invitrogen | 10687010 | |
Ketamine HCL Injection | HENRY SCHEIN ANIMAL HEALTH | 45822 | |
KpnI-HF | NEB | R3142L | |
lenti-CRISPRv2-blast | Addgene | 83480 | |
lenti-Guide-Hygro-iRFP670 | Addgene | 99377 | |
Lipofectamin 3000 Transfection Kit | Invitrogen | L3000015 | |
LV-TRE-VP64-mouse MyoD-T2A-dsRedExpress2 | Addgene | 60625 | |
LV-TRE-VP16 mouse MyoD-T2A-dsRedExpress2 | Addgene | 60626 | |
Mouse on Mouse (M.O.M.) Basic Kit | Vector | BMK-2202 | |
NotI-HF | NEB | R3189L | |
Opti-MEM I Reduced Serum Media | ThermoFisher | 31985070 | |
Polyethylene glycol 4,000 | Alfa Aesar | AAA161510B | |
Polybrene | SIGMA | TR1003 | |
Corning BioCoat Poly-D-Lysine/Laminin Culture Slide | CORNING | CB354688 | |
PowerUp SYBR Green Master Mix | ThermoFisher | A25742 | |
PrimeSTAR Max Premix | TakaRa | R045 | |
Proteinase K | VIAGEN BIOTECH | 507-PKP | |
Puromycin Dihydrochloride | MP Biomedicals | ICN19453980 | |
qPCR Lentivirus Titration Kit | abm | LV900 | |
Quick ligation kit | NEB | M2200S | |
QIAprep Spin Miniprep Kit (250) | QIAGEN | 27106 | |
QIAGEN Plasmid Plus Midi Kit (100) | QIAGEN | 12945 | |
RevertAid RT Reverse Transcription Kit | Thermo scientific | K1691 | |
RNAzol RT | Molecular Research Center, INC | RN 190 | |
T4 DNA Ligase Reaction Buffer | NEB | B0202S | |
T4 Polynucleotide Kinase | NEB | M0201S | |
Terrific Broth Modified | Fisher BioReagents | BP9729-600 | |
ViralBoost Reagent (500x) | ALSTEM | VB100 |