CRISPR/Cas9系统在鸡MSTN上的效率验证

Verification of Efficiency of CRISPR/Cas9 System in Chicken Myostatin Gene

随着基因编辑技术的飞速发展,CRISPR/Cas9技术逐渐成为主流。为了验证CRISPR/Cas9系统在鸡基因组上的切割效率,在鸡肌肉生长抑制素(Myostatin,MSTN)基因第1、第2外显子上各自筛选了5个Cas9靶点,构建p X330-sg RNA表达载体;将GFP质粒电转入DF-1细胞,通过检测表达GFP细胞的比例来优化电转程序;将载体用优化后的电转程序电转入DF-1细胞,培养72 h;最后收集细胞提取基因组,通过T7核酸内切酶Ⅰ(T7EⅠ)酶切试验,选出具有Cas9切割效率的靶点片段,将该片段连入p MD19-T载体,测序并分析结果。结果表明CRISPR/Cas9系统可以成功对DF-1细胞基因组进行切割并引入突变,其中1号外显子上4号靶点的突变率为64.3%,2号外显子上2号靶点的突变率为23.3%。研究表明CRISPR/Cas9系统在DF-1细胞中可以有效切割基因组,为CRISPR/Cas9技术应用于鸡的基因组编辑提供科学依据。

With the rapid development of gene-editing technologies,CRISPR/Cas9 system is gradually becoming the mainstream. This study aimed to verify the targeting efficiency of CRISPR/Cas9 system in chicken genome. We designed5 target sites per exon in exon1 and exon2 of chicken myostatin gene（ch MSTN）,and constructed the corresponding p X330 vectors. Firstly,we transfected DF-1 cells with GFP-contained plasmid using different electrotransfection programs,and chose the most appropriate one whose efficiency was evaluated by ratio of GFP- expressing cells. Secondly,the p X330 vectors were introduced into DF-1 cells by using optimized electrotransfection program respectively. After 72 hours,we detected px330-mediated targeting efficiency of ch MSTN in DF-1 cells by T7 Endonuclease Ⅰ cleavage assay. The DNA fragments contained target sites were linked into p MD19- T vector,and the indel mutations were detected by sanger sequencing. The results showed that CRISPR/Cas9 system could achieve site-specific targeting and introduce mutations into DF-1 cells. The mutation rate of the 4th target site in exon1 was 64.3%,and that of the 2nd target site in exon2 was 23.3%. This study indicated that CRISPR/Cas9 system was efficient in cultured DF-1 cells,and laid a foundation for the application of CRISPR/Cas9 system in chicken genome editing.