title: Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.
abstract: Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency.
authors.affiliation: Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Nakagawa
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan tetsushi-sakuma@hiroshima-u.ac.jp.
authors.forename: Tetsushi
authors.identifier: http://orcid.org/0000-0003-0396-1563
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Cell-Matrix Frontier Laboratory, Health Administration Center, Hiroshima University, 1-2-3 Kasumi, Minamiku, Hiroshima 734-8551, Japan.
authors.forename: Norihisa
authors.initials: N
authors.lastname: Nishimichi
authors.affiliation: Cell-Matrix Frontier Laboratory, Health Administration Center, Hiroshima University, 1-2-3 Kasumi, Minamiku, Hiroshima 734-8551, Japan Clinical Genetics, Hiroshima University Hospital, 1-2-3 Kasumi, Minamiku, Hiroshima 734-8551, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Yokosaki
authors.affiliation: Department of Molecular and Applied Bioscience, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Yanaka
authors.affiliation: Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Takeo
authors.affiliation: Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan.
authors.forename: Naomi
authors.initials: N
authors.lastname: Nakagata
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
keywords: CRISPR-Cas9; Floxed; Inhibin antiserum and equine chorionic gonadotropin (IASe); Knockout; Ultra-superovulation; Zygote