利用CRISPR/Cas9系统和TALEN技术干预XIST基因的表达

Interfering XIST expression by CRISPR/Cas9 and TALEN

XIST是维持雌性哺乳动物X染色体稳定失活的重要长链非编码基因。X染色体失活异常导致X连锁基因的过量表达从而参与了癌症等疾病的发生。干预XIST的表达在XIST的生物学功能和相关疾病发生的研究中是必不可少的。该研究利用CRISPR/Cas9系统和TALEN技术在293T细胞中对已知的XIST核心启动子进行编辑,建立了通过酶切、测序等鉴定突变效率的方法,并且结合极限稀释法、片段分析和TA克隆测序得到基因型确定的XIST低表达的单克隆细胞系。结果显示,CRISPR/Cas9和TALEN对XIST核心启动子的突变可以有效地抑制XIST的表达。该研究表明,针对XIST核心启动子的基因组编辑可以干预XIST的表达,这为长链非编码RNA基因的敲低提供了新的思路。

XIST is one of the long noncoding genes which could help to keep X chromosome silenced in mammals. The abnormity of X chromosome in activation increases the expression of X-linked genes, which is thought to represent a key event in oncogenesis and the occurrence of other diseases. Interfering XIST expression is necessary to study functions of XIST and associated diseases. In this study, we edited the known core promoter of XISTby CRISPR/Cas9 and TALEN in 293T cells. T7E1 assay and sequencing were used to detect the efficiency of mutation. Limiting dilution, fragment analysis and TA cloning were used to get the monoclonal cell lines expressing low XIST and identify their genotypes. Results showed that compared with the control group, both CRISPR/Cas9 and TALEN could mutate the core promoter and knock down the expression of XIST. In conclusion, the edited core promoter of XIST could knock down its expression. The results may contribute to new strategy about silencing long noncoding genes.