Targeting knockout of DMD gene exon51 in HEK293T cell based on CRISPR/Cas9 system
- VernacularTitle:基于CRISPR/Cas9系统的HEK293T细胞DMD基因第51号外显子的靶向敲除
- Author:
Shuang LI
1
;
Shan-Shan MA
;
Si-Ying CUI
;
Su-Zhen QU
;
Ao-Jie CAI
;
Fang-Xia GUAN
;
Xiang-Dong KONG
Author Information
1. 郑州大学第一附属医院 遗传与产前诊断中心
- Keywords:
CRISPR/Cas9;
DMD gene;
single vector plasmid;
HEK293T cell;
exon knockout
- From:
Basic & Clinical Medicine
2018;38(3):375-380
- CountryChina
- Language:Chinese
-
Abstract:
Objective To knockout the exon51 of DMD gene in HEK293T cells using the CRISPR/Cas9 system. Methods Design the target sequences of sgRNA and clone them into plasmid PX459 respectively; transfer these plasmids into HEK293T cell and extract the total genome DNA; test the activity of sgRNAs with surveyor assay, choose the most efficient one in each end;construct plasmid PX459-2sgRNA and transfer it into HEK293T cells;check whether the exon51 has been knocked known with PCR and T vector sequencing. Results 50% of HEK293T cells' DMD gene exon51 were knocked out,showing a high gene editing efficiency. Conclusions We successfully establish a platform to target knockout the exon51 of DMD gene and provide an important experimental basis for the treatment of DMD and other genetic diseases.
