利用CRISPR/Cas9技术构建斑马鱼prkd1基因敲除品系

Construction of the Zebrafish prkd1 Gene Knockout Model Based on CRISPR/Cas9 Technology

摘要

蛋白激酶D1 (protein kinase D1, PKD1; 也称作PRKD1)是蛋白激酶家族成员之一, 该家族由3种结构相关的应激激活酶组成, 可调节机体多种生物学功能, 主要涉及细胞增殖、分化、凋亡、免疫调节、心脏收缩、血管生成和癌症等, 其中PRKD1与心脏肥大、收缩和缺血再灌注损伤的底物磷酸化有关。相关研究报道, 先天性心脏病患者存在PRKD1基因突变, 但其在心脏中的特异性功能和分子机制并未阐明。为了便于后期研究PRKD1基因在人类早期心脏发育的作用机制, 本文拟利用CRISPR/Cas9技术构建斑马鱼prkd1基因敲除品系。首先, 通过生物信息学网站筛选出两个最佳的基因敲除靶位点, 合成相应靶位点的单链向导RNA (single guide RNA, sgRNA)和引物; 然后, 将两个靶位点的sgRNA进行体外转录, 并将其与Cas9蛋白混合后共同注射到斑马鱼的1-细胞期; 最后, 对基因敲除后的F0、F1、F2及F3代斑马鱼的胚胎和成鱼进行有效性鉴定及表型观察。结果显示, 靶位点附近出现了不同程度的碱基缺失; 成功构建了F1代能够稳定遗传的prkd1基因敲除的3个亚系; 与野生型相比, F3代纯合子胚胎表现出不同程度的心腔膨大、环化异常及心管线性化等畸形现象。综上可知, 本研究利用CRISPR/Cas9技术成功构建了斑马鱼prkd1基因敲除品系, 为进一步研究该基因在人类心脏发育中的特异性功能提供了有益参考, 并为后期的先天性心脏病筛查和精准医疗提供了重要依据。

Abstract

Protein kinase D1 (PKD1 or PRKD1) is a member of the protein kinase family, which is composed of three structurally related stress-activating enzymes that regulate a variety of biological functions in the body, mainly involving cell proliferation, differentiation, apoptosis, immune regulation, cardiac contraction, angiogenesis, and cancer. In these processes, PRKD1 is associated with substrate phosphorylation of cardiac hypertrophy, systole, and ischemia-reperfusion injury. Relevant studies have reported the presence of PRKD1 gene mutations in patients with congenital heart disease, but its specific function and molecular mechanism in the heart have not been elucidated. In order to facilitate the study of the mechanism of PRKD1 gene in early human heart development, a zebrafish prkd1 gene knockout line was constructed using CRISPR/Cas9 technology. Firstly, the two optimal gene knockout target sites were screened out through the bioinformatics website, and the single guide RNAs (sgRNAs) and primers of the corresponding target sites were synthesized. Then, the sgRNAs of the two target sites were transcribed in vitro and mixed with Cas9 protein and co-in-jected into the one-cell stage zebrafish embryos. Finally, the zebrafish embryos and adults of the F0, F1, F2 and F3 generations after gene knockout were identified and phenotypically observed. The results showed that there were different base deletions near the target site. Three sublineages of prkd1 gene knockout that can be stably inherited in the F1 generation were successfully constructed. Compared with the wild type, the F3 homozygous embryos showed different degrees of malformations such as cardiac chamber expansion, abnor-mal circularization and cardiac tube linearization. In summary, this study demonstrated the successful con-struction of a zebrafish prkd1 gene knockout line using CRISPR/Cas9 technology, which lays a groundwork for further studying the specific function of this gene in human heart development and is useful for congeni-tal heart disease screening and precision medicine.

关键词

prkd1基因 / CRISPR/Cas9技术 / 基因敲除 / 先天性心脏病(CHD)

Key words

prkd1 gene / CRISPR/Cas9 technology / gene knockout / congenital heart disease (CHD)