Combination of Crispr/cas9 Genome

Editing and iPSCs Technology for

Haematological Disease Therapy 98

Dr. Baoqiang Guo

B.guo@mmu.ac.uk
Learning outcome

By the end of the session you will be able to:

Describe what is genome editing (CRISPR/cas9)

technology.
Describe the pathogenesis of Sickle cell disease
(SCD)
Discuss how iPSCs combine with genome editing to
treat SCD.
 Genetics of SCD

Sickle cell anaemia

(autosomal recessive)

Val HisLeuThrProGluGlu …
1 2 3 4 5 6 7 146
Normal erythrocyte has normal
haemoglobin amino acid sequence
in the beta chain.
Glu6Val in HBB, Glu-hydrophilic(-);
Val, hydrophobic (neutral)

Val His LeuThrProVal Glu …

1 2 3 4 5 6 7 146
Sickled erythrocyte results from a
single amino acid change in the
beta chain of haemoglobin.
Genetics of SCD
 Sickle-cell disease (SCD)
• Excruciating pain in limbs
• Caused by a blocked vessel
• The disease is caused by
mutations in the gene
encoding the hemoglobin
subunit β (HBB).

Normal red blood cells last from 90 to 120 days;

Sickle cells live only 10 to 20 days,
Pathogenesis of SCD
How sickle cell block blood flow
Pathogenesis of SCD

Sundd P et al Annu Rev Pathol. 2019

Clinical features
Clinical features
Clinical features
Clinical features
iPSCs meet CRISPR : Cure sickle cell disease
 The Nobel Prize
in Physiology or Medicine 2012

Reprogramming of human cells:

Mature cells can be reprogrammed to
become pluripotent iPS
1. Disease modelling and drug screening
These groundbreaking discoveries have 2. Replacement therapy.
completely changed our view of the
development and cellular specialisation
 CRISPR genome editing gets 2020
Nobel Prize in Chemistry
Emmanuelle Charpentier and Jennifer A.
Doudna share award

Emmanuelle Charpentier Jennifer A. Doudna

A short video to how
CRISPR/cas9 edit the genes

https://www.youtube.com/w
atch?v=4YKFw2KZA5o
Clustered regularly interspaced short
palindromic repeats (CRISPR)
How Crispr/cas9 system works on
gene editing
Normal iPS cells and replacement therapy

To improve the safety of iPSCs,

modified protocols:
• non-integrating vector:
one containing the(cDNAs) of
Oct3/4, Sox2, and Klf4.
the other containing the c-Myc
cDNA, into mouse embryonic
fibroblasts, creating iPSCs without
evidence of plasmid integration

• using modified synthetic mRNA

• recombinant proteins
Directed differentiation of iPS cells
into HSCs and HPCs

1. EB ( Embroid bodies) formation

methods.
2. Co-culture with Stromal cell line feeder
cells.
3. Feeder-free monolayer differentiation
system.
iPSCs-genome editing technology for
treating Sickle cell disease mouse model
iPS cells grow on X-ray-inactivated
MEF cells
Normal control-iPS derived embroid
body (EB)
 Dysregulated hematopoiesis was found in
NS/JMML by EB colony forming assay (CFU-
assay) and Cytospin-Giemsa staining
EB14-Giemsa staining
CFU-GM BFU-E/CFU-E
Con

Con
A B

NS
NS
NS/JMML

NS/JMML

Colony forming assay showing dysregulated hematopoiesis A: showing that huge

colonies were found in 15-day colonies of Day 14 differentiated EB cells derived from
NS/JMML-iPS cells. B: Giemsa staining of day-14 EB cells differentiated from iPS cell
generated from control, NS and NS/JMML patients.
iPSCs and Genome editing for hereditary
blood disease
Make blood cells with iPSCs technology
Learning outcome

By the end of the session you will be able to:

Describe what is genome editing (CRASPR/cas9)

technology.
Describe the pathogenesis of Sickle cell disease
(SCD)
Discuss how iPSCs combine with genome editing to
treat SCD.
 Reading list

1. Juan J et al Stem Cell Translational Medicine.

2020;9:590–602.

2. Park CY et al Cell Stem Cell 2015

3. Olgasi C. Stem Cell report 2018

Thank you for
attending the lesson