Constructing and identifying of cell line with low-expression miR-374a based on CRISPR/Cas9 system

ObjectiveTo establish the cell line with low-expression miR-374a using CRISPR/CAS9 gene targeting technology.

MethodsThe mature gRNA targeting miR-374a was designed to synthesize the DNA, which was inserted into the CRISPR/CAS9 vector to construct the pSpCas9(BB)-2A-Puro.A549 cells were transfected with the constructed vector, and screened using puromycin.The gene sequences of target site were analyzed using PCR and sequencing method.The qPCR was used to detect the relative expression levels of miR-374a and its target gene TGFA in transfected cells.The CCK8 was used to detect the proliferation activity of cells.

ResultsThe gene targeting miR-374a vector was successfully constructed.The transfected cells were screened under pressure, and the sequencing results showed that the inserting/deletion mutation was found.The results qPCR showed that the expression level of miR-374a significantly decreased, and the expression level of TGFA significantly increased in transfecting gene editing vector compared with control cells(P < 0.01 and P < 0.05).The cell proliferation assay showed that the proliferation activity significantly changed in low-expressed miR-374a cells(P < 0.05 to P < 0.01).

ConclusionsThe low-expressed miR-374a cell line was successfully established, which provides the basis in studying the molecular function.