CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA

Hiromi Miura, Channabasavaiah B Gurumurthy, Takehito Sato, Masahiro Sato, Masato Ohtsuka

Research output: Contribution to journalArticle

41 Citations (Scopus)

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.

Original languageEnglish (US)
Article number12799
JournalScientific reports
Volume5
DOIs
StatePublished - Aug 5 2015

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Clustered Regularly Interspaced Short Palindromic Repeats
Single-Stranded DNA
MicroRNAs
Embryonic Stem Cells
Peptide Elongation Factor 2
Genome
Gene Dosage
Homeobox Genes
RNA Interference
Mutagenesis
Genes
Gene Expression
Injections

ASJC Scopus subject areas

  • General

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CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA. / Miura, Hiromi; Gurumurthy, Channabasavaiah B; Sato, Takehito; Sato, Masahiro; Ohtsuka, Masato.

In: Scientific reports, Vol. 5, 12799, 05.08.2015.

Research output: Contribution to journalArticle

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