Generation of biallelic F0 mutants in medaka using the CRISPR/Cas9 system
Generation of biallelic F0 mutants in medaka using the CRISPR/Cas9 system
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DOI：
10.1111/gtc.12511
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发表时间：
2017-07
期刊：
影响因子：
2.1
通讯作者：
Rie Sawamura;N. Osafune;T. Murakami;Fumiya Furukawa;T. Kitano
中科院分区：
文献类型：
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作者：
Rie Sawamura;N. Osafune;T. Murakami;Fumiya Furukawa;T. Kitano
由于难以在单细胞阶段使用编辑工具，基因组编辑方法生成的几种动物模型在F0代中出现了体细胞镶嵌突变，包括野生型基因组序列。快速产生完全敲除的动物是确定靶基因功能的一大优势。本研究利用CRISPR/Cas9系统研究了F0基因敲除medaka的产生。为了确定该编辑系统是否在单细胞阶段诱导了medaka基因组的突变，将重组Cas9蛋白、tracrRNA和死端crRNA （dnd）注射到转基因olvas - DsRedExpress medaka的单细胞阶段胚胎中，这对生殖细胞发育至关重要。这使得生殖细胞可以通过DsRed荧光可见。从胚胎单细胞期提取的基因组DNA通过测序进行分析。可以预见的是，在注射的胚胎中发现了dnd靶位点的双等位基因突变序列模式。为了研究突变鱼的表型，用鱼苗和成鱼对生殖细胞进行了荧光和组织学观察。突变导致生殖细胞的完全丧失，表明在注射的胚胎中失去了dnd的功能。因此，该系统对于F0基因敲除medaka的生产似乎是非常有效的。
Several animal models generated by genome editing methods develop somatic mosaic mutations including wild‐type genome sequence in F0 generation because it is difficult to use editing tools at the one‐cell stage. Producing complete knockout animals quickly is a great advantage in determining the function of target genes. This study investigated the generation of F0 knockout medaka using the CRISPR/Cas9 system. To determine whether this editing system induced mutations in the medaka genome at the one‐cell stage, recombinant Cas9 protein, tracrRNA and crRNA for dead end (dnd), which is essential for germ cell development, were injected into one‐cell stage embryos of olvas‐DsRedExpress transgenic medaka. This allowed germ cells to be visualized by DsRed fluorescence. Genomic DNA extracted from embryos at the one‐cell stage was analyzed by sequencing. Predictably, biallelic mutated sequence patterns in the target sites of dnd were found in the injected embryos. To investigate the phenotypes of the mutated fish, fluorescent and histological observations of germ cells were carried out using fry and adults. The mutations resulted in a complete loss of germ cells, suggesting loss of function of dnd in the injected embryos. Therefore, this system appears to be extremely effective for the production of F0 knockout medaka.