斑马鱼miR-196a-1基因敲除品系的构建

The Establishment of the Zebrafish miR-196a-1 Knockout Lines

microRNA(miRNA)是一类内生的、长度约为19~23个核苷酸的非编码RNA,通过影响mRNA的稳定性和翻译,来参与基因表达的转录后调控。生物信息学分析表明该基因在各个物种中高度保守。为了阐明该基因在肠道发育中的作用,本文利用Cloning free CRISPR/Cas9基因编辑技术构建miR-196a-1基因敲除的斑马鱼品系。首先通过分析软件筛选出斑马鱼miR-196a-1基因的两个敲除位点,两个敲除位点相隔132 bp,利用PCR技术扩增miR-196a-1的向导DNA,再以向导DNA为模板转录得到miR-196a-1的sgRNA,将miR-196a-1基因的sgRNA和Cas9蛋白共同注射到斑马鱼胚胎1细胞期胚胎中。斑马鱼胚胎发育到36 hpf后进行基因编辑的有效性检测,研究结果显示,miR-196a-1基因出现102 bp碱基的缺失,表明CRISPR/Cas9系统对miR-196a-1基因的敲除有效。对其F0代、F1代、F2代进行筛选,成功获得斑马鱼miR-196a-1基因敲除品系,为研究miR-196a-1在肠道发育中的作用奠定了基础。

MicroRNA(miRNA),a type of endogenous non-coding RNA with a length of about 19~23 nucleotides,participates in post-transcriptional regulation of gene expression by affecting the stability and translation of mRNA.Bioinformatics analysis showed that the gene is highly conserved in various species.In order to study the role of miR-196a-1 gene in intestinal development,we used cloning free CRISPR/Cas9 gene editing technology to establish the zebrafish miR-196a-1 knockout lines.Firstly,two knockout sites of zebrafish miR-196a-1 gene were screened by online analysis,the two knockout sites were 132 bp apart.Next,the template guide DNA of miR-196a-1 gene was amplified by PCR.Then,the template DNA was transcribed into the sgRNA of miR-196a-1 gene.Finally,the sgRNA of miR-196a-1 gene and Cas9 protein were co-injected into zebrafish embryos at the 1-cell stage.The effectiveness of gene editing was tested after 36 hpf.The results showed that there was a deletion of 102 bp base in miR-196a-1 gene,revealing that the CRISPR/Cas9 system is effective in knocking out miR-196a-1 gene.The miR-196a-1 knockout line of zebrafish was established successfully after screening the F0,F1,and F2 generations.These findings laid a foundation for exploring the role of miR-196a-1 in zebrafish intestinal development.