German Center for Neurodegenerative Diseases (DZNE), Schillerstrasse 44, 80336 Munich, Germany.
Development. 2013 Dec;140(24):4982-7. doi: 10.1242/dev.099085. Epub 2013 Nov 20.
Gene modifications in animal models have been greatly facilitated through the application of targeted genome editing tools. The prokaryotic CRISPR/Cas9 type II genome editing system has recently been applied in cell lines and vertebrates. However, we still have very limited information about the efficiency of mutagenesis, germline transmission rates and off-target effects in genomes of model organisms. We now demonstrate that CRISPR/Cas9 mutagenesis in zebrafish is highly efficient, reaching up to 86.0%, and is heritable. The efficiency of the CRISPR/Cas9 system further facilitated the targeted knock-in of a protein tag provided by a donor oligonucleotide with knock-in efficiencies of 3.5-15.6%. Mutation rates at potential off-target sites are only 1.1-2.5%, demonstrating the specificity of the CRISPR/Cas9 system. The ease and efficiency of the CRISPR/Cas9 system with limited off-target effects make it a powerful genome engineering tool for in vivo studies.
通过应用靶向基因组编辑工具，动物模型中的基因修饰得到了极大的促进。原核 CRISPR/Cas9 Ⅱ型基因组编辑系统最近已被应用于细胞系和脊椎动物。然而，我们对模型生物基因组中的突变效率、种系传递率和脱靶效应的了解仍然非常有限。我们现在证明，CRISPR/Cas9 在斑马鱼中的诱变非常高效，达到 86.0%，且可遗传。CRISPR/Cas9 系统的效率进一步促进了供体寡核苷酸提供的蛋白质标签的靶向敲入，敲入效率为 3.5-15.6%。潜在脱靶位点的突变率仅为 1.1-2.5%，表明了 CRISPR/Cas9 系统的特异性。CRISPR/Cas9 系统具有有限的脱靶效应，操作简便且高效，使其成为体内研究中一种强大的基因组工程工具。
