Page 103 - ebook

Development of allele-specific CRISPR gene therapy for
CReVIS-seq: a highly accurate and multiplexable
Epidermolysis Bullosa Simplex Dowling-Meara(EBS-DM)
CRISPR-based method for mapping lentivirus inte-
with single-base precision
Yohan An1, Hyomin Lee2, Junho K Hur1,3,*
Heon Seok Kim1,2,6,†, Gue-Ho Hwang1,2,†, Hyomin Lee3, Taegeun Bae4, Seong-Ho Park3, Yong Jun Kim3,5, Jae-Hoon Park5,
1 Graduate School of Biomedical Science č Engineering, Hanyang University, Seoul, Korea, 2 Department of Medicine, Major in Medical Genetics, Graduate School,
Sun Lee5,*, Sangsu Bae1,2,*, Junho K Hur3,5,*
Hanyang University, Seoul, Korea, 3 Department of Genetics, College of Medicine, Hanyang University, Seoul, Korea
Abstract
Abstract
CRISPR-mediated Frameshft
The Dowling-Meara variant of epidermolysis bullosa simplex
(EBS-DM) is an inherited skin disorder typified by blisters that pri-
marily affect the skin after mechanical trauma. While EBS-DM is a
severe disease that is associated with significant morbidity in in-
fancy and early childhood, effective treatment is currently unavail-
able. The genetic cause of EBS-DM is dominant-negative muta-
tions within either the KRT5 (12q13.13) or KRT14 (17q12.q21)
genes, encoding keratin 5 and keratin 14, respectively. The keratin
proteins are highly abundant in the cytoplasm of basal keratino-
cytes in the epidermis, providing cytoskeletal scaffold within the
epithelial cells for protection against mechanical and non-mechani-
cal stresses. If mutations within either KRT5 or KRT5 genes occur,
keratin 5 and keratin 14 filament network structures become frag-
ile, which leads to epidermal cytolysis and blistering that are symp-
toms of EBS-DM. However, there are currently no therapies ap-
proved for the treatment of EBS-DM, besides symptomatic care. In Figure 1. Scheme of mutant keratin gene targeting by allele-spe-
this regard, gene therapy might be a potential option for corrective cific CRISPR-Cas9
therapy for these patients. To this end, in this study, we used After transfection of reporter vector with sgRNA and Cas9 vector, hu-
CRISPR/Cas9-gRNA to modify mutant KRT5 and KRT14 genes for man cells express RFP, cas9, and sgRNA. Mutant allele-specific Cas-
therapy. We developed allele-specific CRISPR-Cas9 methods to 9/sgRNA don’t cleave wild-type but mutant keratin gene in reporter
target specific mutations associated with EBS-DM. vector. If CRISPR-mediated NHEJ occurs in the keratin gene,
insertion and deletion (Indel) induce frameshift mutation, which
causes GFP gene expression. These cells are analyzed by FACS
Introduction according to RFP and GFP expression.
Skin is the layer of usually soft, flexible outer tissue covering the
body, consisting of epidermis, dermis, and hypodermis. The main Results
functions of the skin are protection, sensation, and heat regulation
that can be abnormally affected by the skin disorder. Epidermolysis Wild type
bullosa (EB) is a group of rare skin disorders that causes the skin to
tear and blister at the slightest touch. Epidermolysis bullosa simplex
(EBS) is responsible for the majority of the subtypes of EB that are
divided by the location of the blister. Also, EBS is divided into the
age of onset and clinical features. Among the subtypes of EBS, the Comp-PerCP-A
Dowling-Meara variant of epidermolysis bullosa simplex (EBS-DM)
is a severe disease that is associated with significant morbidity in
infancy and early childhood. The genetic cause of EBS-DM is
dominant-negative mutations within either the KRT5 (12q13.13) or
KRT14 (17q12.q21) genes, encoding keratin 5 and keratin 14, Mutant
respectively. Mutant keratin 5 and keratin 14 filament network
structures are more fragile than normal structures, which leads to
epidermal cytolysis and blistering that are symptoms of EBS-DM. In
this regard, gene therapy might be a potential option for corrective Comp-PerCP-A
therapy for these patients. To this end, in this study, we used
CRISPR/Cas9-gRNA to modify mutant KRT5 and KRT14 genes for
therapy and developed allele-specific CRISPR-Cas9 with
single-base precision to target specific mutations associated with
EBS-DM.
Overview Comp-GFP-A Comp-GFP-A

Figure 2. Comparison of allele-specific CRISPR/Cas9-mediated
cleavage of wild-type and mutant keratin gene
Transfection with sgRNA and The above results showed that GFP gene was expressed predomi-
Cas9 vector
nantly in the mutant reporter vector, which means that developed
CR- ISPR-Cas9 cleaved specifically mutant reporter gene.

References
References
[1] Fine JD, Johnson LB, Weiner M, Suchindran C. Cause-spe-
cific risks of childhood death in inherited epidermolysis
bullosa. J Pediatr. 2008 Feb;152(2):276-80. doi: 10.1016/j.j-
peds.2007.06.039. Epub 2007 Oct 22. PMID: 18206702.
[2] https://www.debra.org.uk/what-is-eb/about-eb
Mutagenic Non-Homolgous
End Joining (NHEJ)