Chapter 9
Electroporation of CRISPR-Cas9 into Malignant B Cells
for Loss-of-Function Studies of Target Gene Via Knockout
Jian Yu
Abstract
CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit genomic DNA
for studying biology, pathogenesis, and molecular basis of treatment in malignant B cells. Unfortunately,
malignant B cells are extremely difficult to transfect by most traditional methods. In this chapter, we
describe the use of the Nucleofector™ Technology-based electroporation system with optimized transfection conditions for generating a malignant B cell model, JEKO-1, with ROR1-gene knockout via CRISPRCas9 technology.
Key words Electroporation, Nucleofector, CRISPR-Cas9, JEKO-1
1 Introduction
Gene transfer and expression in malignant B cells has increased our
understanding of gene function and is a particularly important tool
for the analysis of the mechanisms leading to malignancy. DNA
transfection studies have allowed the determination of their role in
the progression of carcinogenesis. CRISPR-Cas9 technology has
now become a powerful tool revolutionizing biomedical research
on malignant B cells, due to its capability to knock out target gene
via homology-directed repair (HDR) [1–3]. However, the transfection efficiency of CRISPR-Cas9 into hard-to-transfect malignant
B cells remains to be improved [4]. Successful delivery of sufficient
number of CRISPR/Cas9 elements into malignant B cells by
transfection is a prerequisite for efficient gene editing. Transfection
methods can be broadly classified into viral, chemical, and physical,
among which electroporation is the most widely used physical
method [5]. Electroporation is a procedure that is gaining in popularity by using high-voltage electric shocks to introduce DNA into
cells, which can be used with most hard-to-transfect cell types via
alternate techniques, and yields a high frequency of both stable
Shulin Li et al. (eds.), Electroporation Protocols: Microorganism, Mammalian System, and Nanodevice,
Methods in Molecular Biology, vol. 2050, https://doi.org/10.1007/978-1-4939-9740-4_9,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
85
Electroporation of CRISPR-Cas9 into Malignant B Cells
for Loss-of-Function Studies of Target Gene Via Knockout
Jian Yu
Abstract
CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit genomic DNA
for studying biology, pathogenesis, and molecular basis of treatment in malignant B cells. Unfortunately,
malignant B cells are extremely difficult to transfect by most traditional methods. In this chapter, we
describe the use of the Nucleofector™ Technology-based electroporation system with optimized transfection conditions for generating a malignant B cell model, JEKO-1, with ROR1-gene knockout via CRISPRCas9 technology.
Key words Electroporation, Nucleofector, CRISPR-Cas9, JEKO-1
1 Introduction
Gene transfer and expression in malignant B cells has increased our
understanding of gene function and is a particularly important tool
for the analysis of the mechanisms leading to malignancy. DNA
transfection studies have allowed the determination of their role in
the progression of carcinogenesis. CRISPR-Cas9 technology has
now become a powerful tool revolutionizing biomedical research
on malignant B cells, due to its capability to knock out target gene
via homology-directed repair (HDR) [1–3]. However, the transfection efficiency of CRISPR-Cas9 into hard-to-transfect malignant
B cells remains to be improved [4]. Successful delivery of sufficient
number of CRISPR/Cas9 elements into malignant B cells by
transfection is a prerequisite for efficient gene editing. Transfection
methods can be broadly classified into viral, chemical, and physical,
among which electroporation is the most widely used physical
method [5]. Electroporation is a procedure that is gaining in popularity by using high-voltage electric shocks to introduce DNA into
cells, which can be used with most hard-to-transfect cell types via
alternate techniques, and yields a high frequency of both stable
Shulin Li et al. (eds.), Electroporation Protocols: Microorganism, Mammalian System, and Nanodevice,
Methods in Molecular Biology, vol. 2050, https://doi.org/10.1007/978-1-4939-9740-4_9,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
85