CRISPR Gene Editing in Stem Cells

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Cell Stem Cell
Short Article
A Single CRISPR-Cas9 Deletion Strategy that
Targets the Majority of DMD Patients Restores
Dystrophin Function in hiPSC-Derived Muscle Cells
Courtney S. Young,1,2,3,4 Michael R. Hicks,2,3,5 Natalia V. Ermolova, 2,4 Haruko Nakano,3,7 Majib Jan,2,5,6
Shahab Younesi,2,5,6 Saravanan Karumbayaram,3,5 Chino Kumagai-Cresse,2,4 Derek Wang,2,5 Jerome A. Zack,1,3,5
Donald B. Kohn,1,2,3,5 Atsushi Nakano,1,2,3,7 Stanley F. Nelson,1,2,3,8 M. Carrie Miceli,1,2,3,5 Melissa J. Spencer,1,2,3,4,*
and April D. Pyle1,2,3,5,*
1Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA
2Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA
3Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA
4Department of Neurology, University of California, Los Angeles, CA 90095, USA
5Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
6CIRM Bridges Program, California State University, Northridge, CA 91330, USA
7Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095, USA
8Department of Human Genetics, University of California, Los Angeles, CA 90095, USA
*Correspondence: (M.J.S.), (A.D.P.)
SUMMARY
Mutations in DMD disrupt the reading frame, prevent
dystrophin translation, and causeDuchennemuscular
dystrophy (DMD). Here we describe a CRISPR/Cas9
platformapplicable to 60%of DMDpatient mutations.
We applied the platform to DMD-derived hiPSCs
where successful deletion and non-homologous end
joining of up to 725 kb reframed the DMD gene. This
is the largest CRISPR/Cas9-mediated deletion shown
to date in DMD. Use of hiPSCs allowed evaluation of
dystrophin in disease-relevant cell types. Cardiomyo-
cytes and skeletal muscle myotubes derived from re-
framed hiPSC clonal lines had restored dystrophin
protein. The internally deleted dystrophin was func-
tional as demonstrated by improvedmembrane integ-
rity and restoration of the dystrophin glycoprotein
complex in vitro and in vivo. Furthermore, miR31
was reduced upon reframing, similar to observations
in Becker muscular dystrophy. This work demon-
strates the feasibility of using a single CRISPR pair
to correct the reading frame for the majority of DMD
patients.
INTRODUCTION
Duchenne muscular dystrophy (DMD) is the most common fatal
genetic disease of childhood, affecting 1 in 3,500–5,000 boys.
In DMD, progressive muscle degeneration generally leads to
death in the twenties, and there are currently no highly effective
therapies. DMD is often caused by frameshifting exonic dele-
tions in DMD, which encodes dystrophin. Dystrophin stabilizes
the dystrophin glycoprotein complex (DGC) at the sarcolemma;
loss of functional dystrophin leads to the degradation of DGC
components, which results in muscle membrane fragility and
leakage of creatine kinase (CK) (Pearce et al., 1964). Approxi-
mately 60% of mutations causing DMD occur between DMD
exons 45–55 (Be´ roud et al., 2007). Multiple independent clinical
reports in patients and dystrophic mice have revealed that in-
frame deletions of exons 45–55 produce an internally deleted
dystrophin protein and are associated with a very mild Becker
muscular dystrophy (BMD) disease course, with some patients
still asymptomatic in their sixties (Be´ roud et al., 2007; Echigoya
et al., 2015; Nakamura et al., 2008; Taglia et al., 2015). Thus, ge-
netic manipulation to create a large deletion of exons 45–55 is a
therapeutic strategy to restore the reading frame for 60% of
DMD patients with mutations in this region.
One promising approach to induce genetic correction of
DMD is through the use of the bacterially acquired immune
surveillance system known as clustered regularly interspaced
short palindromic repeats (CRISPR) and CRISPR-associated
nuclease (Cas) 9. In this system a short guide RNA (gRNA), which
is complimentary to a specific site in the genome, is used to
target the Cas9 nuclease and induce double-stranded breaks
(DSBs). The DSBs can be repaired through non-homologous
end joining (NHEJ) or homology-directed repair.
Previous work has shown that CRISPR/Cas9 components can
modify the DMD gene (Li et al., 2015; Long et al., 2014, 2016;
Nelson et al., 2016; Ousterout et al., 2015; Tabebordbar et al.,
2016; Wojtal et al., 2016; Xu et al., 2015). In this investigation,
we describe a therapeutically relevant CRISPR/Cas9 platform
that we designed to modify DMD. Our platform involves excision
of exons 45–55 and NHEJ to reframe dystrophin through crea-
tion of an internally deleted protein that is stable and functional.
The internally deleted protein mimics the naturally occurring
exon 45–55 deletion observed in mild BMD patients and encom-
passes 60% of DMD patient mutations.
For the first time, we demonstrate CRISPR/Cas9-mediated
deletion and NHEJ of up to 725 kb of the DMD gene in human
induced pluripotent stem cell (hiPSC) lines. We show that
CRISPR/Cas9 reframed, hiPSC-derived skeletal and cardiac
Cell Stem Cell 18, 533–540, April 7, 2016 ª2016 Elsevier Inc. 533