electroporation, which enabled genome editing to occur before the
first replication of the mouse genome. The present chapter
describes the method of genome editing by electroporation of
Cas9 protein (GEEP method) to generate genome-edited mice.
2 Materials
2.1 Manipulation
and Culture of Mouse
Embryos
Materials for the manipulation and culture of mouse embryos are
purchased commercially or prepared following the textbook Manipulating the Mouse Embryo: A Laboratory Manual (CSHL press) [8].
1. Pregnant mare serum gonadotropin, PMSG: Dissolve the lyophilized powder hormones at 500 units/ml in sterile distilled
water, and store at À20
C.
2. Human chorionic gonadotropin, hCG: Dissolve the lyophilized powder hormones at 500 units/ml in sterile distilled
water, and store at À20
C.
3. Six female B6D2F1 mice (3–4-week-old).
4. One male B6D2F1 mouse (8–9-week-old).
5. CARD FERTIUP Mouse Sperm Preincubation Medium
(Kyudo Co., Ltd., Saga, Japan).
6. HTF medium.
7. M2 medium.
8. KSOM medium.
9. Mineral oil.
10. Mouth pipette: a glass microcapillary attached to tubing and
aspirator mouthpiece.
2.2 Electroporation
1. Streptococcus pyogenes Cas9 protein containing nuclear localization sequence (NLS) (spCas9): Dissolve 100 μg of spCas9
(IDT, Inc., IL, USA) in 100 μl of Opti-MEM medium. Aliquot
and store at À70
C (see Note 1).
2. CRISPR RNA (crRNA) and trans-activating CRISPR RNA
(tracrRNA): Dissolve crRNA and tracrRNA (IDT, Inc., IL,
USA) in Opti-MEM media at 1 μg/μl. Store at À30
C (see
Note 2).
3. Single-stranded oligodeoxynucleotides (ssODN): Dissolve
ssODN in Opti-MEM medium at 1 μg/μl. Store at À30
C
(see Note 3).
4. Electroporator: CUY21EDIT II or Genome Editor (BEX
Co. Ltd., Tokyo, Japan).
5. Platinum plate electrode: LF501PT1-10 (BEX Co. Ltd.,
Tokyo, Japan).
6. Stereo microscope.
122
Tatsuya Takemoto
first replication of the mouse genome. The present chapter
describes the method of genome editing by electroporation of
Cas9 protein (GEEP method) to generate genome-edited mice.
2 Materials
2.1 Manipulation
and Culture of Mouse
Embryos
Materials for the manipulation and culture of mouse embryos are
purchased commercially or prepared following the textbook Manipulating the Mouse Embryo: A Laboratory Manual (CSHL press) [8].
1. Pregnant mare serum gonadotropin, PMSG: Dissolve the lyophilized powder hormones at 500 units/ml in sterile distilled
water, and store at À20
C.
2. Human chorionic gonadotropin, hCG: Dissolve the lyophilized powder hormones at 500 units/ml in sterile distilled
water, and store at À20
C.
3. Six female B6D2F1 mice (3–4-week-old).
4. One male B6D2F1 mouse (8–9-week-old).
5. CARD FERTIUP Mouse Sperm Preincubation Medium
(Kyudo Co., Ltd., Saga, Japan).
6. HTF medium.
7. M2 medium.
8. KSOM medium.
9. Mineral oil.
10. Mouth pipette: a glass microcapillary attached to tubing and
aspirator mouthpiece.
2.2 Electroporation
1. Streptococcus pyogenes Cas9 protein containing nuclear localization sequence (NLS) (spCas9): Dissolve 100 μg of spCas9
(IDT, Inc., IL, USA) in 100 μl of Opti-MEM medium. Aliquot
and store at À70
C (see Note 1).
2. CRISPR RNA (crRNA) and trans-activating CRISPR RNA
(tracrRNA): Dissolve crRNA and tracrRNA (IDT, Inc., IL,
USA) in Opti-MEM media at 1 μg/μl. Store at À30
C (see
Note 2).
3. Single-stranded oligodeoxynucleotides (ssODN): Dissolve
ssODN in Opti-MEM medium at 1 μg/μl. Store at À30
C
(see Note 3).
4. Electroporator: CUY21EDIT II or Genome Editor (BEX
Co. Ltd., Tokyo, Japan).
5. Platinum plate electrode: LF501PT1-10 (BEX Co. Ltd.,
Tokyo, Japan).
6. Stereo microscope.
122
Tatsuya Takemoto