codon 26 (GAG ! AAG, glutamate to lysine) in another allele (β
E )
resulting in abnormal hemoglobin E production [3]. Allogeneic
hematopoietic stem cell transplantation is the only curative therapy.
However, the treatment is limited by the availability of
HLA-matched donors.
The advent of iPSC technology provides a patient-derived
renewable source of cells amenable to genetic manipulation. Generation of patient-derived iPSCs followed by genetic correction of
mutations and differentiation into specific cell types offer promise
for autologous transplantation. Previously, gene therapy using a
lentiviral vector carrying β-globin (HBB) gene has been reported
in β-thalassemia patient-derived iPSCs [4, 5]. However, this
approach can lead to insertional mutagenesis and therefore requires
the screening of iPSC clones that harbor the HBB transgene at
genomic safe harbor locus.
Recently, the RNA-guided CRISPR/Cas9 system has become
a versatile tool for precise genome editing and been used to efficiently correct the mutations in homozygous β-thalassemia patientderived iPSCs through homology-directed repair (HDR) pathway
[6–9]. In this chapter, we describe the detailed protocol for the
seamless correction of HbE mutation in HbE/β-thalassemia
patient-derived iPSCs (Eβ-iPSCs) using the CRISPR/Cas9 plasmid
and the ssODN repair template according to our previous work
[10]. The gene-targeting strategy involves designing of the gRNA
targeting the point mutation (AAG) and the ssODN template
containing the left homology arm, the correct nucleotide (GAG)
and the right homology arm, and construction and cloning of the
gRNA into the Cas9 expression plasmid (PX459) to obtain the
CRISPR/Cas9 plasmid (sgRNA/PX459). To target the HbE
mutation, we perform nucleofection to deliver both the sgRNA/
PX459 and the ssODN template into the Eβ-iPSCs. The transfected pools are clonally isolated and expanded for verification of
the corrected clones by multiplex PCR analysis and Sanger
sequencing (Fig. 1).
After genetic correction of the HbE mutation in one allele, the
corrected iPSCs become heterozygote. Upon hematopoietic and
erythroid differentiation, the corrected iPSCs can restore HBB
gene and protein expression [10]. This genetic correction strategy
is simpler and more practical than correcting the HBB mutations in
the other allele, which can be heterogeneous with over 200 possible
mutations at the HBB gene being identified.
Therefore, the strategy can be employed as a universal approach
for HbE correction of other types of HbE/β
0 or HbE/β
+
-thalassemia patient-derived iPSCs. This protocol is also applicable to
genome editing of iPSCs for creating small edits such as correction
of point mutations in iPSCs derived from patients with genetic
disorders or introduction of point mutations into the wild-type
iPSCs to create the isogenic diseased iPSC lines for functional
genomics study.
194
Methichit Wattanapanitch
E )
resulting in abnormal hemoglobin E production [3]. Allogeneic
hematopoietic stem cell transplantation is the only curative therapy.
However, the treatment is limited by the availability of
HLA-matched donors.
The advent of iPSC technology provides a patient-derived
renewable source of cells amenable to genetic manipulation. Generation of patient-derived iPSCs followed by genetic correction of
mutations and differentiation into specific cell types offer promise
for autologous transplantation. Previously, gene therapy using a
lentiviral vector carrying β-globin (HBB) gene has been reported
in β-thalassemia patient-derived iPSCs [4, 5]. However, this
approach can lead to insertional mutagenesis and therefore requires
the screening of iPSC clones that harbor the HBB transgene at
genomic safe harbor locus.
Recently, the RNA-guided CRISPR/Cas9 system has become
a versatile tool for precise genome editing and been used to efficiently correct the mutations in homozygous β-thalassemia patientderived iPSCs through homology-directed repair (HDR) pathway
[6–9]. In this chapter, we describe the detailed protocol for the
seamless correction of HbE mutation in HbE/β-thalassemia
patient-derived iPSCs (Eβ-iPSCs) using the CRISPR/Cas9 plasmid
and the ssODN repair template according to our previous work
[10]. The gene-targeting strategy involves designing of the gRNA
targeting the point mutation (AAG) and the ssODN template
containing the left homology arm, the correct nucleotide (GAG)
and the right homology arm, and construction and cloning of the
gRNA into the Cas9 expression plasmid (PX459) to obtain the
CRISPR/Cas9 plasmid (sgRNA/PX459). To target the HbE
mutation, we perform nucleofection to deliver both the sgRNA/
PX459 and the ssODN template into the Eβ-iPSCs. The transfected pools are clonally isolated and expanded for verification of
the corrected clones by multiplex PCR analysis and Sanger
sequencing (Fig. 1).
After genetic correction of the HbE mutation in one allele, the
corrected iPSCs become heterozygote. Upon hematopoietic and
erythroid differentiation, the corrected iPSCs can restore HBB
gene and protein expression [10]. This genetic correction strategy
is simpler and more practical than correcting the HBB mutations in
the other allele, which can be heterogeneous with over 200 possible
mutations at the HBB gene being identified.
Therefore, the strategy can be employed as a universal approach
for HbE correction of other types of HbE/β
0 or HbE/β
+
-thalassemia patient-derived iPSCs. This protocol is also applicable to
genome editing of iPSCs for creating small edits such as correction
of point mutations in iPSCs derived from patients with genetic
disorders or introduction of point mutations into the wild-type
iPSCs to create the isogenic diseased iPSC lines for functional
genomics study.
194
Methichit Wattanapanitch