11. Transfer the zygotes into the culture dish. Place 10 zygotes in
one 20-μl KSOM drop (50 zygotes in one culture dish). Culture the zygotes overnight.
12. Separate two-cell stage embryos from unfertilized oocytes and
damaged zygotes.
13. Transfer 15 two-cell stage embryos into an oviduct of 0.5-dpc
pseudopregnant recipient female mice (30 embryos per one
mouse when the embryo transfer performed bilaterally) (see
Note 8).
4 Notes
1. NLS is essential for genome editing in mouse zygotes.
2. Single guide RNA (sgRNA) can also be used in this protocol.
We prepare sgRNA using in vitro transcription system using
plasmid or PCR amplicon as a template. The concentration of
sgRNA in genome editing solution is the same (100 ng/μl) as
that of crRNA/tracrRNA.
3. No need to purify ssODN with HPLC or SDS-PAGE. Reversephase cartridge purification is enough for ssODN. Purchase
ssODN in dry form, and dissolve in Opti-MEM medium.
4. The higher the concentration of ssODN, the higher the efficiency of HDR-mediated knockin. However, the survival rate
of zygotes decreases in the higher ssODN concentration.
5. To prevent evaporation, fill the electrode with genome editing
solution just before the electroporation.
6. Minimize the amount of carryover of KSOM medium to the
electrode to maintain the concentration of Cas9, crRNA, and
tracrRNA in the genome editing solution.
7. Inbred mouse strains (such as C57BL/6) are sensitive for
electroporation. Therefore, we lower the voltage and the number of pulses to 25 V and 5 pulses, respectively, to ensure the
high survival rate.
8. We recommend transferring 13–15 embryos into an oviduct
because some embryos are damaged by electroporation.
Acknowledgments
We thank members of the Takemoto laboratory and Setsuro
Tech Inc. for support. This work was supported by Grant-in-Aid
for Scientific Research (B) and Grant-in-Aid for Scientific Research
on Innovative Areas “Singularity Biology (No.8007)” for T.T.
Zygote Electroporation for CRISPR/Cas9 Delivery to Generate Genetically. . .
125
one 20-μl KSOM drop (50 zygotes in one culture dish). Culture the zygotes overnight.
12. Separate two-cell stage embryos from unfertilized oocytes and
damaged zygotes.
13. Transfer 15 two-cell stage embryos into an oviduct of 0.5-dpc
pseudopregnant recipient female mice (30 embryos per one
mouse when the embryo transfer performed bilaterally) (see
Note 8).
4 Notes
1. NLS is essential for genome editing in mouse zygotes.
2. Single guide RNA (sgRNA) can also be used in this protocol.
We prepare sgRNA using in vitro transcription system using
plasmid or PCR amplicon as a template. The concentration of
sgRNA in genome editing solution is the same (100 ng/μl) as
that of crRNA/tracrRNA.
3. No need to purify ssODN with HPLC or SDS-PAGE. Reversephase cartridge purification is enough for ssODN. Purchase
ssODN in dry form, and dissolve in Opti-MEM medium.
4. The higher the concentration of ssODN, the higher the efficiency of HDR-mediated knockin. However, the survival rate
of zygotes decreases in the higher ssODN concentration.
5. To prevent evaporation, fill the electrode with genome editing
solution just before the electroporation.
6. Minimize the amount of carryover of KSOM medium to the
electrode to maintain the concentration of Cas9, crRNA, and
tracrRNA in the genome editing solution.
7. Inbred mouse strains (such as C57BL/6) are sensitive for
electroporation. Therefore, we lower the voltage and the number of pulses to 25 V and 5 pulses, respectively, to ensure the
high survival rate.
8. We recommend transferring 13–15 embryos into an oviduct
because some embryos are damaged by electroporation.
Acknowledgments
We thank members of the Takemoto laboratory and Setsuro
Tech Inc. for support. This work was supported by Grant-in-Aid
for Scientific Research (B) and Grant-in-Aid for Scientific Research
on Innovative Areas “Singularity Biology (No.8007)” for T.T.
Zygote Electroporation for CRISPR/Cas9 Delivery to Generate Genetically. . .
125