title: Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes.
abstract: BACKGROUND
  Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing permits the rapid production of genetically engineered mice. To make the most of this innovative technology, a streamlined procedure is needed for the robust construction of CRISPR/Cas9 vectors, the efficient preparation of mouse oocytes, and refined genotyping strategies. Although we previously demonstrated the applicability of oocyte cryopreservation technologies and various genotyping methods in the production of transcription activator-like effector nuclease-mediated genome editing in mice, it has not yet been clarified whether these techniques can be applied to the CRISPR/Cas9-mediated generation of knockout mice. In this study, we investigated easy, efficient, and robust methods of creating knockout mice using several CRISPR/Cas9 systems.
  
  
  RESULTS
  We constructed three types of CRISPR/Cas9 vectors expressing: 1) single guide RNA (gRNA) and Cas9 nuclease, 2) two gRNAs and Cas9 nickase, and 3) two gRNAs and FokI-dCas9, targeting the same genomic locus. These vectors were directly microinjected into the pronucleus of freeze-thawed fertilized oocytes, and surviving oocytes were transferred to pseudopregnant ICR mice. Cas9 nuclease resulted in the highest mutation rates with the lowest birth rates, while Cas9 nickase resulted in the highest birth rates with the lowest mutation rates. FokI-dCas9 presented well-balanced mutation and birth rates. Furthermore, we constructed a single all-in-one FokI-dCas9 vector targeting two different genomic loci, and validated its efficacy by blastocyst analysis, resulting in highly efficient simultaneous targeted mutagenesis.
  
  
  CONCLUSIONS
  Our report offers several choices of researcher-friendly consolidated procedures for making CRISPR/Cas9-mediated knockout mice, with sophisticated construction systems for various types of CRISPR vectors, convenient preparation of in vitro fertilized or mated freeze-thawed oocytes, and an efficient method of mutant screening.
authors.affiliation: Center for Animal Resources and Development, Kumamoto University, Kumamoto, 860-0811, Japan. y-naka@kumamoto-u.ac.jp.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Nakagawa
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, 739-8526, Japan. tetsushi-sakuma@hiroshima-u.ac.jp.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, 739-8526, Japan. m143124@hiroshima-u.ac.jp.
authors.forename: Takuya
authors.initials: T
authors.lastname: Sakamoto
authors.affiliation: Center for Animal Resources and Development, Kumamoto University, Kumamoto, 860-0811, Japan. ohmuraya@kumamoto-u.ac.jp.
authors.forename: Masaki
authors.initials: M
authors.lastname: Ohmuraya
authors.affiliation: Center for Animal Resources and Development, Kumamoto University, Kumamoto, 860-0811, Japan. nakagata@kumamoto-u.ac.jp.
authors.forename: Naomi
authors.initials: N
authors.lastname: Nakagata
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, 739-8526, Japan. tybig@hiroshima-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D005307:Fertilization in Vitro; D051379:Mice; D018345:Mice, Knockout; D008845:Microinjections; D016296:Mutagenesis; D009154:Mutation; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D015053:Zygote
chemical_list: D064130:CRISPR-Associated Proteins; D004247:DNA