transformation and transient gene expression [6]. However, with
the high electric field strength and ensued electrochemical reactions, electroporation often leads to high posttransfection mortality. Moreover, despite the optimization of electrical parameters and
solution recipes, its efficiency on many cell types especially malignant B cells is still not sufficiently high, posing a major obstacle for
its applications [7, 8].
In this chapter, we selected ROR1 as the target gene, which is a
developmentally restricted, type I tyrosine kinase-like orphan
receptor expressed on the neoplastic cells of a variety of different
cancers, including chronic lymphocytic leukemia (CLL) [2] and
mantle cell lymphoma (MCL) [9], but not on most normal postpartum tissues [10]. High-level expression of ROR1 is associated
with accelerated disease progression in patients with CLL and MCL
[2, 9]. On the other hand, silencing ROR1 in CLL cells can
decrease leukemia cell survival [11]. These indicate that ROR1
signaling can promote leukemia cell activation and survival, and
enhance disease progression in patients with B-cell hematologic
malignancies, including CLL and MCL. Knocking out ROR1 by
CRISPR/Cas9 technology might be a new strategy to study the
role of ROR1 in cancer biology and a potential way to treat patients
with ROR1-expressing B-Cell hematologic malignancies. Here, we
report an optimized Nucleofector™ Technology-based electroporation method capable of delivering GFP-expressing plasmid
(pSpCas9(BB)-2A-GFP) bearing both Cas9 and the single-guide
RNA (sgRNA) into hard-to-transfect malignant B cells with a
competent efficiency and a low cytotoxicity. Also, we demonstrate
successful ROR1 knockout study using the CRISPR/Cas9 system
and the electroporation technique and the high HDR rate phenomenon may find broad significant applications.
2 Materials
2.1 Preparation
of DNA
1. The pSpCas9(BB)-2A-GFP plasmid used in this study was
purchased from Addgene (Addgene Inc., Cambridge, MA)
(see Note 1).
2. Custom sgRNA sequence for ROR1 were as follows:
Top: 5
0 -CACCgGCGCTGCTGCTGGCCGCACG-3
0 .
Bottom: 5
0 -AAACCGTGCGGCCAGCAGCAGCGCc-3
0 .
The sgRNA oligos were obtained from IDT (Integrated
DNA Technologies, Inc., San Diego, CA) (see Note 2).
3. Resuspend the top and bottom strands of oligos for the sgRNA
to a final concentration of 100 μM and annealing the sgRNA
oligos after mix.
86
Jian Yu
the high electric field strength and ensued electrochemical reactions, electroporation often leads to high posttransfection mortality. Moreover, despite the optimization of electrical parameters and
solution recipes, its efficiency on many cell types especially malignant B cells is still not sufficiently high, posing a major obstacle for
its applications [7, 8].
In this chapter, we selected ROR1 as the target gene, which is a
developmentally restricted, type I tyrosine kinase-like orphan
receptor expressed on the neoplastic cells of a variety of different
cancers, including chronic lymphocytic leukemia (CLL) [2] and
mantle cell lymphoma (MCL) [9], but not on most normal postpartum tissues [10]. High-level expression of ROR1 is associated
with accelerated disease progression in patients with CLL and MCL
[2, 9]. On the other hand, silencing ROR1 in CLL cells can
decrease leukemia cell survival [11]. These indicate that ROR1
signaling can promote leukemia cell activation and survival, and
enhance disease progression in patients with B-cell hematologic
malignancies, including CLL and MCL. Knocking out ROR1 by
CRISPR/Cas9 technology might be a new strategy to study the
role of ROR1 in cancer biology and a potential way to treat patients
with ROR1-expressing B-Cell hematologic malignancies. Here, we
report an optimized Nucleofector™ Technology-based electroporation method capable of delivering GFP-expressing plasmid
(pSpCas9(BB)-2A-GFP) bearing both Cas9 and the single-guide
RNA (sgRNA) into hard-to-transfect malignant B cells with a
competent efficiency and a low cytotoxicity. Also, we demonstrate
successful ROR1 knockout study using the CRISPR/Cas9 system
and the electroporation technique and the high HDR rate phenomenon may find broad significant applications.
2 Materials
2.1 Preparation
of DNA
1. The pSpCas9(BB)-2A-GFP plasmid used in this study was
purchased from Addgene (Addgene Inc., Cambridge, MA)
(see Note 1).
2. Custom sgRNA sequence for ROR1 were as follows:
Top: 5
0 -CACCgGCGCTGCTGCTGGCCGCACG-3
0 .
Bottom: 5
0 -AAACCGTGCGGCCAGCAGCAGCGCc-3
0 .
The sgRNA oligos were obtained from IDT (Integrated
DNA Technologies, Inc., San Diego, CA) (see Note 2).
3. Resuspend the top and bottom strands of oligos for the sgRNA
to a final concentration of 100 μM and annealing the sgRNA
oligos after mix.
86
Jian Yu