利用CRISPR/Cas9系统敲除斑马鱼bco1基因与bco1l基因
Next-generation Knock-out Technology of bco1 and bco1l Genes in Zebrafish by Using CRISPR/Cas9 System
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摘要: β-胡萝卜素-15,15'-加氧酶(β-carotene-15,15'-momoxygenase 1，bco1)是β-胡萝卜素转化成维生素A过程中的关键酶，bco1与bco1l是编码此酶的主要基因。本实验利用CRISPR/Cas9技术敲除斑马鱼的与β-胡萝卜素-15,15'-加氧酶编码相关的基因bco1与bco1l，以便深入开展对斑马鱼bco1的功能研究。分别在斑马鱼bco1与bco1l基因2号外显子选取sgRNA识别位点，体外转录制备sgRNA并与 Cas9 mRNA 混合对斑马鱼Ⅰ细胞期受精卵进行显微注射，24 h后收集部分胚胎进行PCR检测并将PCR产物进行单克隆测序确定sgRNA的有效性，构建首建鱼，并在此基础上通过PCR检测、凝胶电泳及测序筛选可遗传突变体。本研究分别获得了bco1基因突变与bco1l基因突变，分析表明这些缺失和插入均可导致编码序列的移码，为研究鱼类胡萝卜素代谢及相关发育过程等后续研究提供了材料。
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关键词:
- β-胡萝卜素-15,15'-加氧酶 /
- 斑马鱼 /
- CRISPR/Cas9技术 /
- 基因敲除
Abstract: β-carotene is one of natural precursors of vitamin A, which is indispensable for chordate development. β-carotene-15,15’-oxygenase 1 (bco1) is one of crucial enzymes of vitamin A synthesis processes. bco1 and bco1l are keystone genes related to the bco1 encoding vitamin A synthesis in zebrafish (Danio rerio). As a model organism, zebrafish has been widely used in ecology, evolution, and environment sciences. In the present study, clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system, one kind of genome editing technological approaches, was employed to target the genes to analyze their roles in fish physiology. We used the CRISPR/Cas9 system to knock out bco1 and bco1l in Danio rerio. Results showed that two guide-RNAs of each gene are effective. Screening the mutants by Sanger sequencing, heterozygous and homozygous individuals with heritable genotype were obtained. The knockout zebrafish provide models to explore the roles of β-carotene in embryonic and adult development of Danio rerio.-
Key words:
- β-carotene-15,15'-oxygenase /
- Danio rerio /
- CRISPR/Cas9 system /
- knockout
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