Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
STAR Protoc. 2021 Sep 17;2(4):100785. doi: 10.1016/j.xpro.2021.100785. eCollection 2021 Dec 17.
CRISPR-Cas9-mediated, site-directed mutagenesis in mice generates mosaic founder mice with varied efficiency of desired point mutation and other non-homologous end-joined variants. Here, we present a protocol for design, sample preparation, and analysis for identification of mice with the desired mutation. Deep sequencing provides the proportion of reads of a particular allele for each mouse line. Locked nucleic acid probe-based qPCR provides rapid identification of the mutant allele and can be used for genotyping offspring during subsequent breeding for colony establishment. For complete details on the use and execution of this protocol, please refer to Vasu et al. (2021).
CRISPR-Cas9 介导的小鼠定点突变产生了具有不同期望点突变效率和其他非同源末端连接变体的嵌合起始小鼠。在此，我们提供了一种用于设计、样品制备和分析以鉴定具有所需突变的小鼠的方案。深度测序为每条小鼠品系提供了特定等位基因的reads 比例。基于锁核酸探针的 qPCR 可快速鉴定突变等位基因，并可用于随后的繁殖中鉴定后代的基因型，以建立品系。有关此方案的使用和执行的完整详细信息，请参阅 Vasu 等人（2021 年）。
