Teng Yudong, Arbogast Kelsey, Junge Harald, Chen Zhe
The Genetically Engineered Murine Models Core, Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
Colorado Center for Personalized Medicine Biobank, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
STAR Protoc. 2025 Mar 21;6(1):103543. doi: 10.1016/j.xpro.2024.103543. Epub 2025 Jan 4.
Here, we present a protocol to alter the production of alternatively spliced mRNA variants, without affecting the overall gene expression, through CRISPR-Cas9-engineered genomic mutations in mice. We describe steps for designing guide RNA to direct Cas9 endonuclease to consensus splice sites, producing transgenic mice through pronuclear injection, and screening for desired mutations in cultured mammalian cells using a minigene splicing reporter. Splice isoform-specific mouse mutants provide valuable tools for genetic analyses beyond loss-of-function and transgenic alleles. For complete details on the use and execution of this protocol, please refer to Dailey-Krempel et al. and Johnson et al..
在此，我们展示了一种通过对小鼠进行CRISPR-Cas9基因工程突变来改变可变剪接mRNA变体的产生，而不影响整体基因表达的方案。我们描述了设计引导RNA以将Cas9核酸内切酶导向共有剪接位点的步骤，通过原核注射产生转基因小鼠，以及使用小基因剪接报告基因在培养的哺乳动物细胞中筛选所需突变的步骤。剪接异构体特异性小鼠突变体为功能丧失和转基因等位基因之外的遗传分析提供了有价值的工具。有关此方案的使用和执行的完整详细信息，请参考Dailey-Krempel等人和Johnson等人的研究。
