Carrington Blake, Sood Raman
Zebrafish Core, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
Bio Protoc. 2023 Aug 5;13(15):e4732. doi: 10.21769/BioProtoc.4732.
Generation of zebrafish () models with targeted insertion of epitope tags and point mutations is highly desirable for functional genomics and disease modeling studies. Currently, CRISPR/Cas9-mediated knock-in is the method of choice for insertion of exogeneous sequences by providing a repair template for homology-directed repair (HDR). A major hurdle in generating knock-in models is the labor and cost involved in screening of injected fish to identify the precise knock-in events due to low efficiency of the HDR pathway in zebrafish. Thus, we developed fluorescent PCR-based high-throughput screening methods for precise knock-in of epitope tags and point mutations in zebrafish. Here, we provide a step-by-step guide that describes selection of an active sgRNA near the intended knock-in site, design of single-stranded oligonucleotide (ssODN) templates for HDR, quick validation of somatic knock-in using injected embryos, and screening for germline transmission of precise knock-in events to establish stable lines. Our screening method relies on the size-based separation of all fragments in an amplicon by fluorescent PCR and capillary electrophoresis, thus providing a robust and cost-effective strategy. Although we present the use of this protocol for insertion of epitope tags and point mutations, it can be used for insertion of any small DNA fragments (e.g., LoxP sites, in-frame codons). Furthermore, the screening strategy described here can be used to screen for precise knock-in of small DNA sequences in any model system, as PCR amplification of the target region is its only requirement. Key features This protocol expands the use of fluorescent PCR and CRISPR-STAT for screening of precise knock-in of small insertions and point mutations in zebrafish. Allows validation of selected sgRNA and HDR template within two weeks by somatic knock-in screening. Allows robust screening of point mutations by combining restriction digest with CRISPR-STAT. Graphical overview ).
生成带有表位标签靶向插入和点突变的斑马鱼()模型对于功能基因组学和疾病建模研究非常有必要。目前，CRISPR/Cas9介导的敲入是通过提供同源定向修复(HDR)的修复模板来插入外源序列的首选方法。生成敲入模型的一个主要障碍是由于斑马鱼中HDR途径效率低，筛选注射的鱼以识别精确的敲入事件涉及的劳动力和成本。因此，我们开发了基于荧光PCR的高通量筛选方法，用于在斑马鱼中精确敲入表位标签和点突变。在这里，我们提供了一个逐步指南，描述了在预期敲入位点附近选择活性sgRNA、设计用于HDR的单链寡核苷酸(ssODN)模板、使用注射胚胎快速验证体细胞敲入以及筛选精确敲入事件的种系传递以建立稳定品系。我们的筛选方法依赖于通过荧光PCR和毛细管电泳对扩增子中的所有片段进行基于大小的分离，从而提供一种强大且经济高效的策略。尽管我们展示了该方案用于插入表位标签和点突变，但它可用于插入任何小DNA片段(例如，LoxP位点、框内密码子)。此外，这里描述的筛选策略可用于筛选任何模型系统中小DNA序列的精确敲入，因为对靶区域进行PCR扩增是其唯一要求。关键特性 本方案扩展了荧光PCR和CRISPR-STAT在筛选斑马鱼中小插入和点突变精确敲入方面的应用。通过体细胞敲入筛选，可在两周内验证所选sgRNA和HDR模板。通过将限制性消化与CRISPR-STAT相结合，可对点突变进行强大的筛选。图形概述 )。
