Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing allows for the disruption or modification of genes in a multitude of model organisms. In the present study, we describe and employ the method for use in the fathead minnow (Pimephales promelas), in part, to assist in the development and validation of adverse outcome pathways (AOPs). The gene coding for an enzyme responsible for melanin production, tyrosinase (tyr), was the initial target chosen for development and assessment of the method since its disruption results in abnormal pigmentation, a phenotype obvious within 3-4 d after injection of fathead minnow embryos. Three tyrosinase-targeting guide strands were generated using the fathead minnow sequence in tandem with the CRISPOR guide strand selection tool. The strands targeted two areas: one stretch of sequence in a conserved region that demonstrated homology to EGF-like or laminin-like domains as determined by Protein Basic Local Alignment Search Tool in concert with the Conserved Domain Database, and a second area in the N-terminal region of the tyrosinase domain. To generate one cell embryos, in vitro fertilization was performed, allowing for microinjection of hundreds of developmentally-synchronized embryos with Cas9 proteins complexed to each of the three guide strands. Altered retinal pigmentation was observed in a portion of the tyr guide strand injected population within 3 d post fertilization (dpf). By 14 dpf, fish without skin and swim bladder pigmentation were observed. Among the three guide strands injected, the guide targeting the EGF/laminin-like domain was most effective in generating mutants. CRISPR greatly advances our ability to directly investigate gene function in fathead minnow, allowing for advanced approaches to AOP validation and development.

中文翻译：

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fat鱼（Pimephales promelas）基因CRISPR / Cas9突变的方法。

簇状规则间隔的短回文重复序列（CRISPR）/ Cas9基因组编辑可破坏或修饰多种模型生物中的基因。在本研究中，我们描述并采用了用于黑头min鱼（Pimephales promelas）的方法，部分是为了协助不良结果途径（AOP）的发展和验证。编码负责黑色素产生的酶酪氨酸酶（tyr）的基因是该方法开发和评估的最初目标，因为其破坏会导致色素沉着异常，这种表型在注射黑头min鱼胚胎后3-4天内很明显。使用黑头min鱼序列和CRISPOR引导链选择工具串联生成了三个靶向酪氨酸酶的引导链。这些分支针对两个领域：保守区中的一段序列证明与EGF或层粘连蛋白样结构域具有同源性，这是由蛋白质基本局部比对搜索工具与保守域数据库共同确定的，酪氨酸酶N端区域的第二个区域域。为了产生一个细胞胚胎，进行了体外受精，允许显微注射数百个发育同步的胚胎，其中Cas9蛋白与三条引导链中的每条复合。在施肥后3 d（typ）内，部分tyr引导链注射人群中观察到视网膜色素沉着改变。到14 dpf时，观察到没有皮肤和游泳膀胱色素沉着的鱼。在注入的三个引导链中，靶向EGF / laminin-like结构域的引导最有效地产生突变体。