Ablain Julien, Durand Ellen M, Yang Song, Zhou Yi, Zon Leonard I
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA.
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA.
Dev Cell. 2015 Mar 23;32(6):756-64. doi: 10.1016/j.devcel.2015.01.032. Epub 2015 Mar 5.
CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish.
基因组编辑的CRISPR/Cas9技术极大地促进了在多种生物体中体外和体内基因的靶向失活。在斑马鱼中，通过简单地将引导RNA（gRNA）和Cas9 mRNA注射到单细胞期胚胎中，就可以快速生成基因敲除品系。在此，我们报告一种简单且可扩展的基于CRISPR的载体系统，用于斑马鱼组织特异性基因失活。作为原理验证，我们使用带有驱动Cas9表达的gata1启动子的载体来沉默urod基因，该基因与血红素生物合成有关，特别是在红细胞谱系中。靶向Urod在斑马鱼胚胎中产生了红色荧光红细胞，重现了在yquem突变体中观察到的表型。虽然F0胚胎显示出嵌合基因破坏，但该表型在稳定的F1鱼中表现得非常明显。这种载体系统构成了一种独特的工具，可在空间上控制基因敲除，并极大地拓宽了斑马鱼功能丧失研究的范围。
