Chapter 13
Zygote Electroporation for CRISPR/Cas9 Delivery
to Generate Genetically Modified Mice
Tatsuya Takemoto
Abstract
The CRISPR/Cas9 system is a powerful tool for generation of genetically modified mice. In conventional
protocols, Cas9 protein (or mRNA) and sgRNA are introduced into zygotes by microinjection. However,
microinjection requires special skill and is too time-consuming to treat zygotes on a large scale. Recently, we
have developed a simple electroporation method which generates genetically modified mice with high
efficiency. Here, we describe our method GEEP (genome editing by electroporation of Cas9 protein). This
method facilitates high-throughput genetic analysis of the mouse. This chapter describes the GEEP method
to generate genetically modified mice.
Key words Genome editing, CRISPR/Cas9, Zygote electroporation
1 Introduction
Genome editing is a powerful tool for elucidating the role of genes
or the genome in various biological processes [1]. It also facilitates
genetic modifications in various species. In particular, since the
development of the CRISPR/Cas9 system, genome editing technique has been widely used in many laboratories [2, 3]. To generate
genome edited mice by CRISPR/Cas9, Cas9 protein or mRNA,
crRNA, and tracrRNA (or sgRNA) have to be delivered into
zygotes. Microinjection is one of the conventional methods for
the delivery of the CRISPR/Cas9 components [4]. However, it
requires a high level of manipulation skill and takes a lot of time to
treat zygotes. To circumvent those issues, we have recently developed a simple electroporation method to introduce the CRISPR/
Cas9 system into mouse zygotes and generated genome-edited
mice with high efficiency [5–7]. In this method, we introduced
Cas9 protein and sgRNA into in vitro fertilized zygotes by
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_13,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
121
Zygote Electroporation for CRISPR/Cas9 Delivery
to Generate Genetically Modified Mice
Tatsuya Takemoto
Abstract
The CRISPR/Cas9 system is a powerful tool for generation of genetically modified mice. In conventional
protocols, Cas9 protein (or mRNA) and sgRNA are introduced into zygotes by microinjection. However,
microinjection requires special skill and is too time-consuming to treat zygotes on a large scale. Recently, we
have developed a simple electroporation method which generates genetically modified mice with high
efficiency. Here, we describe our method GEEP (genome editing by electroporation of Cas9 protein). This
method facilitates high-throughput genetic analysis of the mouse. This chapter describes the GEEP method
to generate genetically modified mice.
Key words Genome editing, CRISPR/Cas9, Zygote electroporation
1 Introduction
Genome editing is a powerful tool for elucidating the role of genes
or the genome in various biological processes [1]. It also facilitates
genetic modifications in various species. In particular, since the
development of the CRISPR/Cas9 system, genome editing technique has been widely used in many laboratories [2, 3]. To generate
genome edited mice by CRISPR/Cas9, Cas9 protein or mRNA,
crRNA, and tracrRNA (or sgRNA) have to be delivered into
zygotes. Microinjection is one of the conventional methods for
the delivery of the CRISPR/Cas9 components [4]. However, it
requires a high level of manipulation skill and takes a lot of time to
treat zygotes. To circumvent those issues, we have recently developed a simple electroporation method to introduce the CRISPR/
Cas9 system into mouse zygotes and generated genome-edited
mice with high efficiency [5–7]. In this method, we introduced
Cas9 protein and sgRNA into in vitro fertilized zygotes by
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_13,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
121