title: Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering.
abstract: The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency.
authors.affiliation: 1] Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan [2].
authors.forename: Takuro
authors.initials: T
authors.lastname: Horii
authors.affiliation: 1] Division of Developmental Biotechnology, Department of Bioscience and Genetics Research Institute, National Cerebral and Cardiovascular Center, 5-7-1 Fujishiro-dai, Suita Osaka 565-8565, Japan [2].
authors.forename: Yuji
authors.initials: Y
authors.lastname: Arai
authors.affiliation: 1] Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan [2] Department of Laboratory Sciences, Graduate School of Health Sciences, Gunma University, 3-39-22 Showa-machi, Maebashi, Gunma 371-8514, Japan [3] Department of Obstetrics and Gynecology, Gunma CHUO General Hospital, 1-7-13, Kouun-cho, Maebashi, Gunma 371-0025, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Yamazaki
authors.affiliation: Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan.
authors.forename: Sumiyo
authors.initials: S
authors.lastname: Morita
authors.affiliation: Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Kimura
authors.affiliation: Department of Obstetrics and Gynecology, Gunma CHUO General Hospital, 1-7-13, Kouun-cho, Maebashi, Gunma 371-0025, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Itoh
authors.affiliation: Department of Laboratory Sciences, Graduate School of Health Sciences, Gunma University, 3-39-22 Showa-machi, Maebashi, Gunma 371-8514, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Abe
authors.affiliation: Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan.
authors.forename: Izuho
authors.initials: I
authors.lastname: Hatada
mesh_terms: D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D016678:Genome; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008845:Microinjections; D008969:Molecular Sequence Data; D009154:Mutation; D015203:Reproducibility of Results; D016415:Sequence Alignment