title: CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA.
abstract: Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Miura
authors.affiliation: Mouse Genome Engineering Core Facility, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
authors.forename: Channabasavaiah B
authors.initials: CB
authors.lastname: Gurumurthy
authors.affiliation: Department of Immunology, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
authors.forename: Takehito
authors.initials: T
authors.lastname: Sato
authors.affiliation: Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8544, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.affiliation: 1] Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan [2] The Institute of Medical Sciences, Tokai University, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004277:DNA, Single-Stranded / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics; D005260:Female; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007438:Introns; D008297:Male; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000235:genetics*; D008845:Microinjections; D016254:Mutagenesis, Insertional; D051857:Otx Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D034622:RNA Interference
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D035683:MicroRNAs; D051857:Otx Transcription Factors; C497660:Otx2 protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000076987:CRISPR-Associated Protein 9; C000600049:Cas9 protein, Francisella novicida; D004720:Endonucleases