文章基本信息

标题：CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA
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作者：Hiromi Miura ; Channabasavaiah B Gurumurthy ; Takehito Sato 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2015
卷号：5
期号：1
DOI：10.1038/srep12799
语种：English
出版社：Springer Nature
摘要：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 C lustered R egularly I nterspaced S hort P alindromic R epeats/ C rispr as sociated 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.