De novo screening of disease-resistant genes from the chromosome-level genome of rare minnow using CRISPR-cas9 random mutation.
De novo screening of disease-resistant genes from the chromosome-level genome of rare minnow using CRISPR-cas9 random mutation.
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DOI：
10.1093/gigascience/giab075
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发表时间：
2021-11-19
期刊：
影响因子：
9.2
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Huang R;Shi M;Luo L;Yang C;Ou M;Zhang W;Liao L;Li Y;Xia XQ;Zhu Z;Wang Y
突变体对于功能基因的发现和种质资源的创造具有重要意义。突变体的获得取决于突变技术和筛选方法的效率。CRISPR-Cas9技术是一种高效的基因编辑技术，主要用于编辑少数基因或靶位点，尚未应用于随机突变体文库的构建和功能基因的从头发现。本研究首先对作为出血症易感模型的野生型稀有鲫（Gobiocypris rarus）的染色体水平基因组进行测序和组装，获得了956.05 Mb的基因组序列，将序列组装成25条染色体，并注释了26，861个蛋白编码基因。之后，应用CRISPR-Cas9技术，以启动子和编码区的保守碱基（TATAWAW和ATG）为靶位点，对稀有米诺鱼的全基因组进行随机突变。罕见的出血性疾病的存活率从0%（整个野生型种群在感染后死亡）逐渐增加到38.24%（F3代）。通过基因组比较分析和细胞水平验证，最终确定了7个敏感基因。结果提供了野生型稀有米诺鱼的基因组资源，并证实本研究利用CRISPR-Cas9技术设计的随机突变系统简单高效，适合于功能基因的从头发现和质量性状相关种质资源的创建。
Mutants are important for the discovery of functional genes and creation of germplasm resources. Mutant acquisition depends on the efficiency of mutation technology and screening methods. CRISPR-Cas9 technology is an efficient gene editing technology mainly used for editing a few genes or target sites, which has not been applied for the construction of random mutant libraries and for the de novo discovery of functional genes. In this study, we first sequenced and assembled the chromosome-level genome of wild-type rare minnow (Gobiocypris rarus) as a susceptible model of hemorrhagic disease, obtained a 956.05 Mb genome sequence, assembled the sequence into 25 chromosomes, and annotated 26,861 protein-coding genes. Thereafter, CRISPR-Cas9 technology was applied to randomly mutate the whole genome of rare minnow with the conserved bases (TATAWAW and ATG) of the promoter and coding regions as the target sites. The survival rate of hemorrhagic disease in the rare minnow gradually increased from 0% (the entire wild-type population died after infection) to 38.24% (F3 generation). Finally, 7 susceptible genes were identified via genome comparative analysis and cell-level verification based on the rare minnow genome. The results provided the genomic resources for wild-type rare minnow, and confirmed that the random mutation system designed using CRISPR-Cas9 technology in this study is simple and efficient and is suitable for the de novo discovery of functional genes and creation of a germplasm resource related to qualitative traits.
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