CRISPR Gene Editing in Stem Cells

deletion that encompasses the majority of DMD mutations,

this approach is optimized for future clinical studies. It would

be unreasonable to design, validate, and evaluate off targets

for every new CRISPR pair tailored for each individual patient.

Additionally, CRISPR/Cas9 is advantageous over exon skip-

ping, as it results in permanent restoration of the reading

frame as opposed to transient effects on RNA splicing. Previ-

ously, Li et al. (2015) used CRISPR/Cas9 to induce exon skip-

ping, frameshifting, or exon knockin to restore dystrophin in a

DMD hiPSC line with an exon 44 deletion; however, their plat-

form is only applicable to 3%–9% of DMD patients (Bladen

et al., 2015), and two of their strategies relied on the creation

of indels, which would be difficult to apply consistently to

each patient. While Ousterout et al. deleted exons 45–55,

they removed significantly less of the intervening region

(336 kb) and thus their approach would cover fewer patient

mutations within the hotspot region. This is because many

mutations extend into the intronic region; thus, by designing

gRNAs that encompass more of the intron, our platform is

applicable to more patients.

Another benefit of using this platform to delete a large portion

DMD

, as opposed to single exons, is the known correlation of

DYS

45–55

with a mild BMD phenotype. Large deletions in the rod

domain of dystrophin often produce a more functional (more like

wild-type) protein, than even very small deletions (Harper et al.,

2002). Larger deletions, which remove hinge III (exons 50–51),

are believed to lead to a milder BMD phenotype than smaller de-

letions, or those that retain hinge III (Carsana et al., 2005). Thus,

in many cases larger deletions are more therapeutically benefi-

cial than smaller ones, due to the way they affect the secondary

structure of the protein.

In summary, we have developed a potentially therapeutic

gene editing platform for DMD to permanently restore the dys-

trophin reading frame in multiple patient-derived hiPSCs. Our

approach using CRISPR/Cas9 and NHEJ deletes up to 725

kb of

DMD

encompassing exons 45–55 and restores dystro-

phin protein function in both cardiomyocytes and skeletal mus-

cle cells derived from reframed hiPSCs. A current limitation of

this platform is that clinical protocols still need to be developed

that allow rapid clonal line derivation and the utilization of

hiPSC-derived cardiac and skeletal muscle progenitors com-

bined with gene correction. Alternatively, CRISPR/Cas9 to

restore the reading frame in DMD mouse models has been

delivered directly in vivo (Long et al., 2016; Nelson et al.,

2016; Tabebordbar et al., 2016). Thus, applications of this plat-

form in the future will allow for the development of an in situ

gene strategy or ex vivo gene correction followed by autolo-

gous cell transplantation, either of which offers tremendous po-

tential for DMD.

EXPERIMENTAL PROCEDURES

Differentiation of hiPSCs to Skeletal Muscle Cells and

Cardiomyocytes

Skeletal muscle differentiation from hiPSCs was induced using OE of a tamox-

ifen inducible MyoD-ERT lentivirus or an adapted 50 day directed differentia-

tion protocol where NCAM

HNK1 cells underwent fluorescence-activated

cell sorting at day 50. Cardiomyocytes were derived through aggregates

over 30 days. See Supplemental Experimental Procedures.

Engraftment into Immunodeficient Mice

NSG immunodeficient mice (Jackson Laboratory) were crossed to mdx

scid

mice (Jackson Laboratory) to generate NSG-mdx mice (see Supplemental

Experimental Procedures). Five- to seven-week-old NSG-mdx mice were

pretreated with 50

l of 10

M cardiotoxin (Sigma-Aldrich) injected into the

right TA 24 hr prior to engraftment. For MyoD OE cells, 100

l of 5 mg/ml

tamoxifen (Sigma-Aldrich) was i.p. injected for 5 days beginning on the day

prior to engraftment. 1

cells in HBSS were injected intramuscularly

and the TA was harvested after 30 days. See Supplemental Experimental

Procedures.

Hypo-osmotic Stress CK Release Assay

Terminally differentiated skeletal muscle cells and cardiomyocytes plated in

duplicate were stressed by incubation in hypo-osomolar solutions ranging

from 66 to 240 mosmol (see Supplemental Experimental Procedures) for

20 min at 37 C. CK was measured in triplicate from the supernatant and cell

lysate with the Creatine Kinase-SL kit (Sekisui Diagnostics) according to the

manufacturer’s instructions.

SUPPLEMENTAL INFORMATION

Supplemental Information for this article includes four figures, two tables and

Supplemental Experimental Procedures and can be found with this article on-

line at

http://dx.doi.org/10.1016/j.stem.2016.01.021.

AUTHOR CONTRIBUTIONS

Conceptualization, Methodology, Writing–Original Draft, Visualization, Proj-

ect Administration, C.S.Y., M.J.S., and A.D.P.; Validation, C.S.Y., M.R.H.,

and N.V.E.; Formal Analysis, C.S.Y.; Investigation, C.S.Y., M.R.H., N.V.E.,

H.N., M.J., S.Y., S.K., C.K.-C., and D.W.; Resources, A.N., S.F.N., M.C.M.,

M.J.S., and A.D.P.; Writing–Review and Editing, C.S.Y., M.R.H., N.E.V.,

S.K., C.K.-C., D.B.K., A.N., S.F.N., M.C.M., M.J.S., and A.D.P.; Supervision,

J.A.Z., D.B.K., A.N., M.C.M., M.J.S., and A.D.P.; Funding Acquisition, M.J.S.

and A.D.P.

(B) Fold change in expression of miR31 measured by ddPCR in myotubes derived from out-of-frame or reframed hiPSCs by MyoD OE, normalized to wild-type

(CDMD 1002). Data are presented as average ± SD.

-dystroglycan. Extracts were from out-of-frame and reframed skeletal muscle myotubes derived by MyoD OE.

HSMM was used as a positive control. Samples were also probed with anti-MyHC as a loading control (bottom panel).

(D) Immunocytochemical staining of MyHC (red) and

-dystroglycan (green), a component of the DGC, in wild-type (CDMD 1002), out-of-frame (CDMD 1006), or

reframed (CDMD 1006-1) skeletal muscle myotubes. Inset depicts zoomed-in region defined by the white box. Scale bar, 50

(E) Assessment of human dystrophin restoration in wild-type (CDMD 1002), out-of-frame (CDMD 1003), and reframed (CDMD 1003-49) MyoD OE cells engrafted

into the TA of NSG-mdx mice. Engrafted human cells were identified by co-immunostaining for human spectrin and lamin A/C (shown in red). Positive staining for

human dystrophin is shown in green and all fibers are shown using laminin (gray). All sections were stained with DAPI (blue) to identify nuclei. Scale bar, 100

(F) Assessment of

-dystroglycan restoration in human fibers from wild-type (CDMD 1002), out-of-frame (CDMD 1003), and reframed (CDMD 1003-49) MyoD OE

cells engrafted into the TA of NSG-mdx mice. Engrafted human cells were identified by co-immunostaining for human spectrin and lamin A/C (shown in red).

Positive staining for dystrophin is shown in gray and

-dystroglycan is shown in green. All sections were stained with DAPI (blue) to identify nuclei. Cell order is the

same as noted in (E). Scale bar, 20