State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Innovation Academy of Seed Design, Chinese Academy of Sciences, Wuhan, China.
University of Chinese Academy of Sciences, Beijing, China.
Methods Mol Biol. 2023;2545:491-512. doi: 10.1007/978-1-0716-2561-3_26.
Research on the evolutionary fate of duplicated genes in recurrent polyploids is scarce due to the difficulties in disentangling the different homeologs and alleles of duplicated genes. This chapter describes the detailed procedures to identify different homeologs and alleles of duplicated genes, to analyze their molecular characteristics, and to reveal their functional divergence by gene editing with CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9). Using the gene editing approach, we efficiently constructed multiple knockout mutant lines with single or simultaneously disrupted different homeologs or alleles in a recurrent polyploid fish, demonstrating its usability for targeting and mutating multiple divergent homeologs and alleles in recurrent duplicated genomes.
由于难以区分重复基因的不同同源基因和等位基因，因此对在反复出现的多倍体中重复基因的进化命运的研究很少。本章描述了鉴定重复基因的不同同源基因和等位基因、分析其分子特征以及通过 CRISPR/Cas9（成簇规律间隔短回文重复序列/CRISPR 相关系统 9）进行基因编辑来揭示其功能分化的详细过程。使用基因编辑方法，我们在反复出现的多倍体鱼类中高效地构建了多个具有单个或同时破坏不同同源基因或等位基因的敲除突变体系，证明了该方法可用于靶向和突变反复出现的重复基因组中的多个不同同源基因和等位基因。
