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

Summary

Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmd^mdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system.

Abstract

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 Dmd^mdx 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.

Introduction

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 degeneration^1,2. 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 sustai....

Protocol

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 Dmd^mdx mice

1. Euthanize adult Dmd^mdx mice (male, 2 months old) by CO₂ asphyxiation and thoracotomy according to IACUC approved by Medical College of Georgia, Augusta University. Cut the tail with a sterile scalpel in a sterile condition under the laminar hood. Rinse the tail with 70% ethanol for 5 min, and then wash with sterile phosphate-b....

Results

Establishment of Dmd^mdx skin fibroblasts derived iPSC. We demonstrated the efficiency of generating mouse iPSCs from Dmd^mdx 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 s.......

Discussion

Duchenne Muscular Dystrophy (DMD) is a destructive and ultimately fatal hereditary disease characterized by a lack of dystrophin, leading to progressive muscle atrophy^1,2. Our results demonstrate the restored dystrophin gene expression in Dmd^mdx iPSC-derived myogenic progenitor cells by the approach of CRISPR/Cas9-mediated exon23 deletion. This approach has three advantages.

First, we generated iPSCs from Dmd^mdx m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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