Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States.
Recombinetics, Inc, St. Paul, United States.
Elife. 2020 May 15;9:e53968. doi: 10.7554/eLife.53968.
Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22-100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.
高效精确的基因组工程需要在基因组靶位点处具有高频率和特异性的整合。在这里，我们描述了一组资源，可简化斑马鱼报告基因敲入，并展示该方法在哺乳动物细胞中的更广泛应用。我们的方法使用 24-48bp 的短同源性，通过同源介导的末端连接（HMEJ）在靶基因的双链断裂处以高频率驱动 DNA 报告基因盒的靶向整合。我们的载体系列 pGTag（用于基因标记的质粒）包含由通用 CRISPR sgRNA 序列侧翼的报告基因，这使得同源臂能够在体内释放。我们在八个斑马鱼基因座观察到靶向敲入的高生殖系传递率（22-100%），并且在猪和人细胞中的安全港基因座有效整合。我们的系统为 CRISPR 和 TALEN 兼容系统中的高效基因靶向应用提供了一种简单且具有成本效益的方法。
