Duchenne muscular dystrophy (DMD) is a genetic disorder caused by mutations in the dystrophin gene. This study used CRISPR/Cas9 genome editing to correct a duplication in exon 2 of the DMD gene in myoblast cells from two DMD patients. A single guide RNA was designed to target the duplicated intronic region. Transfection of the Cas9 plasmid and guide RNA resulted in expression of dystrophin protein in one patient's cells but not the other, possibly due to their different duplication sizes. This demonstrates the potential of CRISPR/Cas9 to treat DMD caused by duplications but also highlights challenges in universal application due to genetic variability between patients.

1. CRISPR/Cas9 for the Correction
of Duchenne Muscular
Dystrophy
(Correction of the Exon 2 Duplication in DMD Myoblasts
by a single CRISPR/Cas9 System)
Nofia Fira Sagrang (0340946)
Yvonne Lie (0338389)
Sonny Roy (0340859)
Yee Zhen Yong (0334953)

2. ● DMD is an inherited muscular disease that involves
muscle weakness, which quickly gets worse.
● DMD is caused by a genetic mutation in the dystrophin (a
protein in the muscles) gene on the X chromosome.
● The symptoms of DMD are progressive weakness and loss
(atrophy) of skeletal and heart muscles. In its early stages,
DMD affects the shoulder and upper arm muscles as well
as the hip and thigh muscles
What is DMD?
Fig. 1: A muscles made up of bundles of fibers (cells). A
group of interdependent proteins along the membrane
surrounding each fiber helps to keep muscle cells working
properly.

3. DMD Exon 2 Duplications
The corresponding gRNA named according to the repeated region, cr
(CRISPR), DMD (Disease name), In (intron), Ex (exon), and numbered
based on the respective notation.
Fig. 2: Diagram representing the 10 kb of the minimal common duplicated region and the position and orientation of the gRNAs (orange)
designed with ZiFiT website in respect to the exon 2 of the DMD gene (green)

4. ● Stands for “Clustered Regularly Interspaced Short Palindromic Repeats”
● Genome-editing tool that enables genetics and researchers to edit part of
the genome by removing, adding, or altering sections of the DNA
sequence.
● The simplest, most flexible, and precise method of genetic manipulation.
● Consist of two main components:
1. Cas9 RNA-guided nuclease
2. Guide RNA (gRNA)
● Able to remove the most frequent duplication in the DMD gene.
What is Crispr/Cas9

5. Experimental design
DNA collection
Dna collected by MLPA
and Immortalized
myoblast from 2 patient.
gRNA Design and Off-Target
Prediction
Designed based on the most
common duplicated region
among DMD patients.
gRNA cloned into MLM3636
backbone.
Cell Cultures and
Transfection
Transfected HEK293T cells
with the Cas9 plasmid.
Transfection of spCas9 and
DMD gRNAs
T7EI (T7 endonuclease 1)
analysis
PCR amplicons for targeted
and off-target genomic
regions
Detection of Deletions and Inversions
Prepare two specific PCRs with a
common forward primer and a second
forward primer.
Inversions in cells with duplication of the
exon 2
Expression analysis
Rna and protein
production

6. Result
● VCN (Vector Copy Distribution)
analysis after 72 hour of
transduction
● Patient 1 (#994), transfer rate >
40% for all type of LV.CRISPR
● Patient 2 (#515), transfer rate
no more than 19%, for all type
of LV.CRISPR
Table. 1: VCN (Vector Copy Distribution) analysis after 72 hour of transduction,indicating the #515 has low
LV.CRISPR transfer in every gRNA scoring no more than 19% for LV_ex2.1 gRNA, compared with the #994
immortalized patient cells that have more than 40% transfer rate for LV_ex2.1 and LV_int2.6, 45% for
LV_int2.1

7. Western Blotting Immunocytochemistry
● Patient 1 (#994), has expression on Dystrophin
and Cas9 protein for all the gRNA.
● The expression of Dystrophin among Patient 1 cell
lines were not as intense as the wild-type cell lines
● Patient 2 (#515), barely express Cas9 protein,
although the Dystrophin were not detected by the
antibodies.
● Patient 1 (#994). Immunocytochemistry analysis
towards dystrophin concentration in LV_DMDin2.1,
and LV_DMDin2.6.
● The LV_DMDin2.1 express more dystrophin
compared with LV_DMDin2.6.
● Although the expression of dystrophin in
LV_DMDin2.1 is not as intense as the wild-type
version.
Fig. 4: Western blotting showing the expression of Dystrophin from #994 (patient 1)
has similar intensity as the control wild-type cells, #515 (patient 2) transformed cells
line did not show any Dystrophin expression
Fig. 5: Immunocytochemistry assay showing the detailed expression of
dystrophin from crDMDint2.1 gRNA in cultured myotubes transfected cells.

8. Discussion
Gene Knockout using lentiviral-vectors were successful based
on the result showing positive expression of spCas9 and
dystrophin.
The incapability of patient 2 (#515) cells line to transformed effectively
may caused by the larger duplication (263 kb) compared with patient 1
(#994) cells line (137 kb). Hence, limiting the transduction efficiency.
Indicating the treatment for DMD patients is not applicable
universally, meaning the diversity of mutation has to be
observed thoroughly per person.

9. Challenges
● Best gRNA is produced by not detecting any off-target activity.
● AAV (Adeno-associated Virus) viral genome are lost in the regenerating
dystrophic skeletal muscle.
This could mimic a transient expression of the CRISPR/Cas9 system,
which reduce the risk of immune response and off-target activity.

10. Current Development
Further Research
● Adeno-associated virus (AAV) are
considered as better vector for in vivo
model.
● In Zhang et al (2020) the expression of
dystrophin is 80% in EDL muscle of
DMD injected ∆Ex44 mice animal
model
AAV Vector
● Amongst DMD patients, intronic
duplication in exon 2 is the most
common causes, deletion towards
introns was highly suggested.
● Increasing in editing efficiency, and
reducing the off-target effects.
Optimal
Target Gene
● Gene-editing using CRISPR was
indicating positive result, hence
further research may opened the
elimination of gene related diseases.

11. Conclusion
DMD is a fatal X-linked recessive neuromuscular disease as a result of mutations that interrupt
the reading frame of the dystrophin gene (DMD). CRISPR/Cas9 technology has gather interest
as a method for DMD therapy due to its potential for permanent exon skipping, which can
restore the disrupted DMD reading frame in DMD and lead to dystrophin restoration. The
removal of a duplication event by Cas9-mediated genome editing can be achieve with only one
gRNA directed against a duplicated intronic region, which is increase the editing efficiency and
reducing the risk of off-target effects. These prove that a gene-editing approach in DMD
patients carrying duplications and give foundation for further research into the application
such an approach in other inherited disorders caused by gene duplications.

12. References
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