Stolfi Alberto, Gandhi Shashank, Salek Farhana, Christiaen Lionel
Center for Developmental Genetics, Department of Biology, New York University, New York, NY 10003, USA
Center for Developmental Genetics, Department of Biology, New York University, New York, NY 10003, USA.
Development. 2014 Nov;141(21):4115-20. doi: 10.1242/dev.114488.
The CRISPR/Cas9 system has ushered in a new era of targeted genetic manipulations. Here, we report the use of CRISPR/Cas9 to induce double-stranded breaks in the genome of the sea squirt Ciona intestinalis. We use electroporation to deliver CRISPR/Cas9 components for tissue-specific disruption of the Ebf (Collier/Olf/EBF) gene in hundreds of synchronized Ciona embryos. Phenotyping of transfected embryos in the 'F0' generation revealed that endogenous Ebf function is required for specification of Islet-expressing motor ganglion neurons and atrial siphon muscles. We demonstrate that CRISPR/Cas9 is sufficiently effective and specific to generate large numbers of embryos carrying mutations in a targeted gene of interest, which should allow for rapid screening of gene function in Ciona.
CRISPR/Cas9系统开创了靶向基因操作的新时代。在此，我们报告了使用CRISPR/Cas9在海鞘（Ciona intestinalis）基因组中诱导双链断裂的情况。我们利用电穿孔法将CRISPR/Cas9组件导入数百个同步化的海鞘胚胎中，以实现对Ebf（Collier/Olf/EBF）基因的组织特异性破坏。对“F0”代转染胚胎的表型分析表明，内源性Ebf功能对于表达胰岛的运动神经节神经元和心房虹吸管肌肉的特化是必需的。我们证明，CRISPR/Cas9足够有效且具有特异性，能够产生大量在感兴趣的靶向基因中携带突变的胚胎，这将有助于在海鞘中快速筛选基因功能。
