Gene engineering has attracted worldwide attention because of its ability of precise location of disease mutations in genome. As a new gene editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system is simple, fast, and accurate to operate at a specific gene site. It overcomes the long-standing problem of conventional operation. At the same time, stem cells are a good foundation for establishing disease model in vitro. Therefore, it has great significance to combine stem cells with the rapidly developing gene manipulation techniques. In this review, we mainly focus on the mechanism of CRISPR/Cas9 technology and its application in stem cell genomic editing, so as to pave the way for promoting rapid application and development of CRISPR/Cas9 technology.

Objective:To investigate the effect of long non-coding RNA (LncRNA) LY6E-DT on temozolomide (TMZ) resistance in high grade glioma cells (HGG) cells and its mechanism.Methods:Bioinformatics screened the LncRNA correlated to the expression of O6-methylguanine-DNA methyltransferase (MGMT) in HGG tissue, and excavated potentially related microRNA (miRNA) . HGG cell U251 was cultured and randomly divided into control group, resistance group and interference group. Resistance group and interference group were treated with 0.2-16.0 μg·ml -1 TMZ gradient incremental induction, and isosmotic PBS was used to treat control group cells. Control group and resistance group were transfected with control vector, while the interference group was transfected with shRNA interference vector targeting LY6E-DT by lentivirus. The semi-inhibitory concentration (IC 50) of TMZ in each group was detected by CCK-8 experiment, the expression levels of LY6E-DT, miR-125a-5p and MGMT mRNA in each group were detected by real-time fluorescent quantitative PCR (RT-qPCR) , and the expression levels of MGMT protein were detected by Western blot. The effect of miR-125a-5p on MGMT and LY6E-DT was detected by luciferase reporter gene method. Results:LY6E-DT expression was positively correlated with MGMT (Pearson correlation coefficient = 0.32, P<0.001) , and it was found that LY6E-DT expression was significantly lower in HGG tissues, but it was not conducive to the prognosis of HGG patients; In the control group, resistance group and interference group, the expressions of LY6E-DT were 1.000±0.047, 3.704±0.402 and 0.743±0.064; the expressions of miR-125a-5p were 1.000±0.049, 0.351±0.031 and 0.934±0.050; the expressions of MGMT mRNA were 1.000±0.017, 5.205±0.462 and 3.183±0.667; the expressions of MGMT protein are 0.108±0.012, 0.891±0.063 and 0.375±0.038; TMZ IC 50 are 6.79±0.71, 30.52±3.69 and 15.64±2.25 μg·ml -1. Compared with the control group, LY6E-DT, MGMT and TMZ IC50 in the resistance group were significantly higher, while LY6E-DT expression in the interference group was significantly lower, MGMT and TMZ IC 50 were significantly higher ( P<0.05) ; Compared with the resistance group, the expression of LY6E-DT, MGMT mRNA and protein and TMZ IC 50 decreased significantly in the interference group ( P<0.05) . miR-125a-5p significantly inhibited luciferase expression of MGMT 3'UTR ( P<0.01) , while LY6E-DT significantly restored the expression level ( P<0.01) . Conclusion:LY6E-DT can promote MGMT expression and TMZ resistance in HGG cells, which is related to the inhibition of miR-125a-5p expression.