Tyrosinase (TYR) converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA into L-dopaquinone, which can produce melanin pigment. The abrogation of the functional activity of TYR can result in albino skin and eye diseases because of a deficiency in melanin pigment production. In this study, we developed and characterized an inducible knockout TYR platform comprising the heat-inducible heat-shock-promoter-70-driving CRISPR/Cas9 system and a zU6-promoter-driving tyr single guide RNA (sgRNA) system to investigate the temporal expression of TYR genes. To overcome the difficulty of identifying zebrafish germline integrations and facilitate the observation of Cas9 expression, heart-specific cmlc2:enhanced green fluorescent protein (EGFP; used to confirm tyr sgRNA expression) and two selectable markers (P2A-mCherry and internal ribosomal entry site-EGFP) were applied in our system. Heat shock treatment administered to Cas9 transgenic embryos induced mCherry or EGFP fluorescence expression throughout the embryos' bodies, and Cas9 protein was detected 1 h after heat shock treatment. Mutations were created by direct injection and line crossing, which led to mosaic and complete depigmentation phenotypes in approximately 50% and 100% of the embryos, respectively. Using our system, conditional TYR knockout in zebrafish was achieved efficiently and simply.

中文翻译：

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CRISPR在斑马鱼中热休克诱导的酪氨酸酶基因消融。

酪氨酸酶（TYR）将L-酪氨酸转化为3,4-二羟基苯丙氨酸（L-DOPA），L-DOPA转化为L-多巴醌，可产生黑色素。TYR功能活性的取消可能会由于黑色素色素生成不足而导致白化病皮肤和眼睛疾病。在这项研究中，我们开发并表征了诱导型敲除TYR平台，该平台包括可热诱导的热休克启动子70驱动CRISPR / Cas9系统和zU6启动子驱动的轮胎单向导RNA（sgRNA）系统，以研究时间序列TYR基因的表达。为了克服识别斑马鱼种系整合的困难并促进Cas9表达的观察，心脏特异性cmlc2：增强型绿色荧光蛋白（EGFP; 用于确认tyr sgRNA的表达）和两个选择标记（P2A-mCherry和内部核糖体进入位点-EGFP）被应用到我们的系统中。对Cas9转基因胚胎进行热休克处理后，整个胚胎体内均会诱导mCherry或EGFP荧光表达，并且在热休克处理后1小时检测到Cas9蛋白。通过直接注射和线交产生突变，分别导致大约50％和100％的胚胎发生镶嵌和完全脱色表型。使用我们的系统，斑马鱼中有条件的TYR基因敲除得以有效而简单地实现。热激处理1 h后检测到Cas9蛋白。通过直接注射和线交产生突变，分别导致大约50％和100％的胚胎发生镶嵌和完全脱色表型。使用我们的系统，斑马鱼中有条件的TYR基因敲除得以有效而简单地实现。热激处理1 h后检测到Cas9蛋白。通过直接注射和线交产生突变，分别导致大约50％和100％的胚胎发生镶嵌和完全脱色表型。使用我们的系统，可以高效，简单地实现斑马鱼的有条件TYR基因敲除。