i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
STAR Protoc. 2020 Dec 10;1(3):100208. doi: 10.1016/j.xpro.2020.100208. eCollection 2020 Dec 18.
Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. For complete details on the use and execution of this protocol, please refer to Amorim et al. (2020).
自首次应用于定点突变以来，CRISPR-Cas9 系统彻底改变了基因组工程。在此，我们提供了一种在斑马鱼中进行靶向基因组缺失的验证工作流程，包括单向导 RNA 和 Cas9 mRNA 的设计、克隆和合成，然后在斑马鱼胚胎中进行显微注射，并进行基因型筛选以建立突变系。该流水线的多功能性和效率使得斑马鱼模型的产生成为功能遗传学中广泛使用的方法。有关该方案使用和执行的完整详细信息，请参阅 Amorim 等人。(2020)。
