Tang and Weintraub were partially supported by NIH-AR070029, NIH-HL086555, NIH-HL134354.

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The authors have nothing to disclose.

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&#8211;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.

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 (&#181;-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&#160;Dmdmdx mouse, a mouse model for DMD, has a point mutation in exon 23 of the&#160;dystrophin&#160;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.

<table><tbody><tr><td>Surgical Instruments</td><td></td><td></td><td></td></tr><tr><td>31-gauge needle</td><td>Various&amp;nbsp;</td><td></td><td></td></tr><tr><td>Sharp Incision</td><td>Various&amp;nbsp;</td><td></td><td></td></tr><tr><td>Sterile Scalpels</td><td>Various&amp;nbsp;</td><td></td><td></td></tr><tr><td>Tweezers</td><td>Various&amp;nbsp;</td><td></td><td></td></tr><tr><td>Fibroblast medium (for 100 mL of complete medium)</td><td>Company</td><td>Catalog Number</td><td>Volume</td></tr><tr><td>2-Mercaptoethanol (55 mM)</td><td>Gibco</td><td>21-985-023</td><td>0.1 mL</td></tr><tr><td>Antibiotic Antimycotic Slution 100x</td><td>CORNING</td><td>MT30004CI</td><td>1 mL</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium - high glucose</td><td>SIGMA</td><td>D6429</td><td>87 mL</td></tr><tr><td>Fetal Bovine Serum Characterized</td><td>HyClone</td><td>SH30396.03</td><td>10 mL</td></tr><tr><td>L-Glutamine solution</td><td>SIGMA</td><td>G7513</td><td>1 mL</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)&amp;nbsp;</td><td>Gibco</td><td>11140076</td><td>1 mL</td></tr><tr><td>TVP solution (for 500 mL of complete solution)</td><td>Company</td><td>Catalog Number</td><td>Volume</td></tr><tr><td>Chicken Serum</td><td>Gibco</td><td>16110-082</td><td>5 mL</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758</td><td>186 mg</td></tr><tr><td>Phosphat-buffered saline</td><td></td><td></td><td>to 500 mL</td></tr><tr><td>Trypsin (2.5%)</td><td>Thermo</td><td>15090046</td><td>5 mL</td></tr><tr><td>mES growth medium(for 500 mL of complete solution)</td><td>Company</td><td>Catalog Number</td><td>Volume</td></tr><tr><td>2-Mercaptoethanol (55 mM)</td><td>Gibco</td><td>21-985-023</td><td>0.5 mL</td></tr><tr><td>Antibiotic Antimycotic Slution 100x</td><td>CORNING</td><td>MT30004CI</td><td>5 mL</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium - high glucose</td><td>SIGMA</td><td>D6429</td><td>408.5 mL</td></tr><tr><td>Fetal Bovine Serum Characterized</td><td>HyClone</td><td>SH30396.03</td><td>75 mL</td></tr><tr><td>L-Glutamine solution</td><td>SIGMA</td><td>G7513</td><td>5 mL</td></tr><tr><td>Mouse recombinant Leukemia Inhibitory Factor (LIF), 0.5 x 106 U/mL</td><td>EMD Millipore Corp</td><td>CS210511</td><td>500 &amp;mu;L</td></tr><tr><td>MEK/GS3 Inhibitor Supplement</td><td>EMD Millipore Corp</td><td>CS210510-500UL</td><td>500 &amp;mu;L</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)&amp;nbsp;</td><td>Gibco</td><td>11140076</td><td>5 mL</td></tr><tr><td>The ES cell media should not be stored for more than 4 weeks and with inhibitors not more than 2 weeks.</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;mES frozen medium(for 50 mL of complete solution)&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Volume&lt;/strong&gt;</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>SIGMA</td><td>D2650</td><td>5 mL</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium - high glucose</td><td>SIGMA</td><td>D6429</td><td>24.9 mL</td></tr><tr><td>Fetal Bovine Serum Characterized</td><td>HyClone</td><td>SH30396.03</td><td>25 mL</td></tr><tr><td>Mouse recombinant Leukemia Inhibitory Factor (LIF), 0.5 x 106 U/mL</td><td>EMD Millipore Corp</td><td>CS210511</td><td>50 &amp;mu;L</td></tr><tr><td>Name of Material/ Equipment</td><td>Company</td><td>Catalog Number</td><td>RRID</td></tr><tr><td>0.05% Trypsin/0.53 mM EDTA</td><td>CORNING</td><td>25-052-CI</td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Thermo scientific</td><td>J19943-k2</td><td></td></tr><tr><td>Accutase solution</td><td>SIGMA</td><td>A6964</td><td>Cell detachment solution</td></tr><tr><td>AgeI-HF</td><td>NEB</td><td>R3552L</td><td></td></tr><tr><td>Alexa488-conjugated goat-anti-mouse antibody</td><td>Invitrogen</td><td>A32723</td><td>AB_2633275</td></tr><tr><td>Alexa488-conjugated goat-anti-rabbit antibody</td><td>Invitrogen</td><td>A32731</td><td>AB_2633280</td></tr><tr><td>Alexa555-conjugated goat-anti-rabbit antibody</td><td>Invitrogen</td><td>A32732</td><td>AB_2633281</td></tr><tr><td>anti-AFP</td><td>Thermo scientific</td><td>RB-365-A1</td><td>AB_59574</td></tr><tr><td>anti-&amp;alpha;-Smooth Muscle Actin (D4K9N) XP</td><td>CST</td><td>19245S</td><td>AB_2734735</td></tr><tr><td>anti-Dystrophin</td><td>Thermo</td><td>PA5-32388</td><td>AB_2549858</td></tr><tr><td>anti-LIN28A (D1A1A) XP&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>CST</td><td>8641S</td><td>AB_10997528</td></tr><tr><td>anti-MYH2</td><td>DSHB</td><td>mAb2F7</td><td>AB_1157865</td></tr><tr><td>anti-Nanog-XP</td><td>CST</td><td>8822S</td><td>AB_11217637</td></tr><tr><td>anti-Oct-4A (D6C8T)</td><td>CST</td><td>83932S</td><td>AB_2721046</td></tr><tr><td>anti-Sox2</td><td>abcam</td><td>ab97959</td><td>AB_2341193</td></tr><tr><td>anti-SSEA1(MC480)&amp;nbsp;</td><td>CST</td><td>4744s</td><td>AB_1264258</td></tr><tr><td>anti-TH (H-196)</td><td>SANTA CRUZ&amp;nbsp;</td><td>sc-14007</td><td>AB_671397</td></tr><tr><td>Alkaline Phosphatase Live Stain (500x)</td><td>Thermo</td><td>A14353</td><td></td></tr><tr><td>Blasticidin S</td><td>Sigma-Aldrich</td><td>203350</td><td></td></tr><tr><td>BsmBI/Esp3I</td><td>NEB</td><td>R0580L/R0734L</td><td></td></tr><tr><td>Carbenicillin</td><td>Millipore</td><td>205805-250MG</td><td></td></tr><tr><td>Collagenase IV&amp;nbsp;</td><td>Worthington Biochemical Corporation</td><td>LS004189</td><td></td></tr><tr><td>Competent Cells</td><td>TakaRa</td><td>636763</td><td></td></tr><tr><td>CutSmart&amp;nbsp;</td><td>NEB</td><td>B7204S</td><td></td></tr><tr><td>CytoTune-iPS 2.0 Sendai Reprogramming Kit</td><td>Thermo</td><td>A16517</td><td></td></tr><tr><td>DirectPCR Lysis Reagent (cell)</td><td>VIAGEN BIOTECH</td><td>302-C</td><td></td></tr><tr><td>Dispase (1 U/mL)</td><td>STEMCELL Technologies</td><td>7923</td><td></td></tr><tr><td>Doxycycline Hydrochloride</td><td>Fisher BioReagents</td><td>BP26535</td><td></td></tr><tr><td>EcoRI-HF</td><td>NEB</td><td>R3101L</td><td></td></tr><tr><td>Fibronectin bovine plasma</td><td>SIGMA</td><td>F1141</td><td></td></tr><tr><td>&amp;nbsp;&lt;br/&gt; QIAEX II Gel Extraction Kit (500)</td><td>QIAGEN</td><td>20051</td><td></td></tr><tr><td>Gelatin from porcine skin, type A</td><td>SIGMA</td><td>G1890</td><td></td></tr><tr><td>HardSet Antifade Mounting Medium with DAPI</td><td>Vector</td><td>H-1500</td><td></td></tr><tr><td>Hygromycin B (50 mg/mL)</td><td>Invitrogen</td><td>10687010</td><td></td></tr><tr><td>Ketamine HCL Injection</td><td>HENRY SCHEIN ANIMAL HEALTH</td><td>45822</td><td></td></tr><tr><td>KpnI-HF</td><td>NEB</td><td>R3142L</td><td></td></tr><tr><td>lenti-CRISPRv2-blast</td><td>Addgene</td><td>83480</td><td></td></tr><tr><td>lenti-Guide-Hygro-iRFP670</td><td>Addgene</td><td>99377</td><td></td></tr><tr><td>Lipofectamin 3000 Transfection Kit</td><td>Invitrogen</td><td>L3000015</td><td></td></tr><tr><td>LV-TRE-VP64-mouse MyoD-T2A-dsRedExpress2&amp;nbsp;&amp;nbsp;</td><td>Addgene</td><td>60625</td><td></td></tr><tr><td>LV-TRE-VP16 mouse MyoD-T2A-dsRedExpress2</td><td>Addgene</td><td>60626</td><td></td></tr><tr><td>Mouse on Mouse (M.O.M.) Basic Kit</td><td>Vector</td><td>BMK-2202</td><td></td></tr><tr><td>NotI-HF</td><td>NEB</td><td>R3189L</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Media</td><td>ThermoFisher</td><td>31985070</td><td></td></tr><tr><td>Polyethylene glycol 4,000</td><td>Alfa Aesar</td><td>AAA161510B</td><td></td></tr><tr><td>Polybrene</td><td>SIGMA</td><td>TR1003</td><td></td></tr><tr><td>Corning&amp;nbsp;BioCoat Poly-D-Lysine/Laminin Culture Slide</td><td>CORNING</td><td>CB354688</td><td></td></tr><tr><td>PowerUp SYBR Green Master Mix</td><td>ThermoFisher</td><td>A25742</td><td></td></tr><tr><td>PrimeSTAR Max Premix</td><td>TakaRa</td><td>R045</td><td></td></tr><tr><td>Proteinase K</td><td>VIAGEN BIOTECH</td><td>507-PKP</td><td></td></tr><tr><td>Puromycin Dihydrochloride</td><td>MP Biomedicals</td><td>ICN19453980</td><td></td></tr><tr><td>qPCR Lentivirus Titration Kit</td><td>abm</td><td>LV900</td><td></td></tr><tr><td>Quick ligation kit</td><td>NEB</td><td>M2200S</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit (250)</td><td>QIAGEN</td><td>27106</td><td></td></tr><tr><td>QIAGEN Plasmid Plus Midi Kit (100)</td><td>QIAGEN</td><td>12945</td><td></td></tr><tr><td>RevertAid RT Reverse Transcription Kit</td><td>Thermo scientific</td><td>K1691</td><td></td></tr><tr><td>RNAzol RT</td><td>Molecular Research Center, INC</td><td>RN 190</td><td></td></tr><tr><td>T4 DNA Ligase Reaction Buffer</td><td>NEB</td><td>B0202S</td><td></td></tr><tr><td>T4 Polynucleotide Kinase</td><td>NEB</td><td>M0201S</td><td></td></tr><tr><td>Terrific Broth Modified</td><td>Fisher BioReagents</td><td>BP9729-600</td><td></td></tr><tr><td>ViralBoost Reagent (500x)</td><td>ALSTEM</td><td>VB100</td><td></td></tr></tbody></table>

crispr/cas9 technology in restoring dystrophin expression in ipsc-derived muscle progenitors

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.

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&#160;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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59432/59432fig01.jpg" /> 
Figure 1: Reprogramming skin fibroblasts from Dmdmdx mice into iPSCs. 
(A) Representative image of ES-like colonies (scale bar = 200 &#181;m). (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 &#181;m). (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 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59432/59432fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59432/59432fig02.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59432/59432fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59432/59432fig03.jpg" /> 
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 &#60; 0.05 vs D0, D6, D10, #P &#60; 0.05 vs D0, D3, D10, $P &#60; 0.05 vs D0, D3, D6, n = 4 for Oct4) (*P &#60; 0.05 vs D6 and D10, #P &#60; 0.05 vs D0, D3, and D10, $P &#60; 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 &#181;m). Right: Immunofluorescent analysis of MYH2 in myotube formation from Dox-induced mouse iPSCs (scale bar = 20 &#181;m). (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 &#60; 0.0001, n = 3). (D) Immunofluorescent analysis of dystrophin expression in Dox-induced MPC from iPSCCas9-Ctrl and iPSCCas9-gRNA (scale bar = 50 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59432/59432fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Guide primers</td>
		</tr>
		<tr>
			<td>i22 sense</td>
			<td>5&#39;-CACCGTTAAGCTTAGGTAAAATCAA- 3&#39;</td>
		</tr>
		<tr>
			<td>i22 antisense</td>
			<td>5&#8217;-AAACTTGATTTTACCTAAGCTTAAC-3&#8217;</td>
		</tr>
		<tr>
			<td>i23 sense</td>
			<td>5&#39;-CACCGAGTAATGTGTCATACCTTCT- 3&#39;</td>
		</tr>
		<tr>
			<td>I23 antisense</td>
			<td>5&#8217;-AAACAGAAGGTATGACACATTACTC-3&#8217;</td>
		</tr>
		<tr>
			<td colspan="2">PCR primers</td>
		</tr>
		<tr>
			<td>OCT4-Forward</td>
			<td>5&#39;-AGCTGCTGAAGCAGAAGAGGATCA-3&#39;</td>
		</tr>
		<tr>
			<td>OCT4-Reverse</td>
			<td>5&#39;-TCTCATTGTTGTCGGCTTCCTCCA-3&#39;</td>
		</tr>
		<tr>
			<td>ACTA1-Forward</td>
			<td>5&#39;-GATCCATGAGACCACCTACAAC-3&#39;</td>
		</tr>
		<tr>
			<td>ACTA1-Reverse</td>
			<td>5&#39;-TCAGCGATACCAGGGTACAT-3&#39;</td>
		</tr>
		<tr>
			<td>Exon22-Forward</td>
			<td>5&#39;-TTACCACCAATGCGCTATCA-3&#39;</td>
		</tr>
		<tr>
			<td>Exon22-Reverse</td>
			<td>5&#39;-CCGAGTCTCTCCTCCATTATTTC-3&#39;</td>
		</tr>
		<tr>
			<td>Exon23-Forward</td>
			<td>5&#39;-CCAAGAAAGCACCTTCAGAAATATG-3&#39;</td>
		</tr>
		<tr>
			<td>Exon23-Reverse</td>
			<td>5&#39;-TTTGGCAGCTTTCCACCA-3&#39;</td>
		</tr>
		<tr>
			<td>Exon24-Forward</td>
			<td>5&#39;-AAC CTT ACA GAA ATG GAT GGC-3&#39;</td>
		</tr>
		<tr>
			<td>Exon24-Reverse</td>
			<td>5&#39;-TTTCAGGATTTCAGCATCCC-3&#39;</td>
		</tr>
		<tr>
			<td>GAPDH-Forward</td>
			<td>5&#39;-TGACAAGCTTCCCATTCTCG-3&#39;</td>
		</tr>
		<tr>
			<td>GAPDH-Reverse</td>
			<td>5&#39;-CCCTTCATTGACCTCAACTACAT-3&#39;</td>
		</tr>
	</tbody>
</table>
Table 1: Primer sequence.

Watch this Scientific Journal Video about CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors at JoVE.com

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

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 ...

crisprcas9-technology-restoring-dystrophin-expression-ipsc-derived

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.3K Views.  Augusta University. Duchenne is een spierdystrofie veroorzaakt voortijdige uitputten van spiervererver voorloper cellen. Het is een van de meest ernstige spierdystrofies. Om een functionele spierregeneratie te bevorderen, gebruiken we CRISPR/Cas9-gemedieerde genbewerking om dystrofineexpressie te herstellen in geïnduceerde pluripotente spiervererverstoorercellen.De CRISPR-Cas9 genbewerking heeft het potentieel om dystrofisch genexpressie te herstellen. Autologe iPSC-afgeleide spiervererveroorzaker cellen...

Video: CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

Genetics

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Bioengineering

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, and as the disease progresses, the patient develops symptoms of dilated cardiomyopathy, arrhythmia, and congestive heart failure. The DMDMDX mutant mice do not express dystrophin, and are commonly used as a mouse model of DMD. In our recent study, we observed that intramyocardial injection of wide type (WT)-myogenic progenitor cells-derived exosomes (MPC-Exo) transiently restored the expression of dystrophin in the myocardium of DMDMDX mutant mice, which was associated with a transient improvement in cardiac function suggesting that WT-MPC-Exo may provide an option to relieve the cardiac symptoms of DMD. This article describes the technique of MPC-Exo purification and transplantation into hearts of DMDMDX mutant mice.

Here, we present a protocol to transiently improve cardiac function in Duchenne muscular dystrophy mice by transplanting exosomes derived from normal myogenic progenitor cells.

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, specifically to study the function of new genes/proteins associated with a disease, identified during the genetic diagnosis. For the development of such cell lines, two major issues have to be untangled: insertion of the CRISPR tools (the Cas9 and the guide RNA) with high efficiency into the chosen cells, and restriction of the Cas9 activity to the specific deletion of the chosen gene. The protocol described here is dedicated to the insertion of the CRISPR tools in difficult to transfect cells, such as muscle cells. This protocol is based on the use of lentiviruses, produced with plasmids publicly available, for which all the cloning steps are described to target a gene of interest. The control of Cas9 activity has been performed using an adaptation of a previously described system called KamiCas9, in which the transduction of the cells with a lentivirus encoding a guide RNA targeting the Cas9 allows the progressive abolition of Cas9 expression. This protocol has been applied to the development of a RYR1-knock out human muscle cell line, which has been further characterized at the protein and functional level, to confirm the knockout of this important calcium channel involved in muscle intracellular calcium release and in excitation-contraction coupling. The procedure described here can easily be applied to other genes in muscle cells or in other difficult to transfect cells and produce valuable tools to study these genes in human cells.

The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out clones.

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Medicine

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

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