The Study Of Up-regulating IL-2 Expression In NK Cells By Using CRISPR/Cas9 Genome Editing Technique

Objectives To edit RBBP4 gene in HEPG2 cell line by using CRISPR/Cas9 genome editing technique.To investigate the effect of knockout in RBBP4 gene after editing and the genome editing technique.To explore the genome knockout technique at the AAVS1 locus in Peripheral blood of humans NK cells and the expression of IL-2 in Peripheral blood of humans NK cells after genome editing.Methods The sgRNAs which targeted exon 1 of RBBP4 gene were designed and subcloned into the GRNA-CAS9 vector.The recombined plasmids were verified by sequencing and transfected into HEPG2 cell line.The genomic RNA and protein of the new monoclonal cell lines were extracted.The effect knockout of RBBP4 gene in cell line by QPCR and Western bloting.To extract NK cells from human peripheral blood mononuclear cells(PBMC).The sgRNAs which targeted gene were designed and plasmid vector were synthesized.The NK cells were cultured and transfection with sgRNAs and plasmid vector.The expression of IL-2 in NK cells were observed by QPCR and ELISA.Results The gene and protein expression of RBBP4 in HEPG2 cell line were significantly reduced than those in wild type cell line.The gene and protein expression of IL-2 in NK cells were significantly increased after CRISPR/Cas9 genome editing.Conclusions CRISPR/Cas9n plasmids that targeted RBBP4 is successfully constructed therefore a RBBP4 knockout RBBP4 stable cell line is established via this system.CRISPR/Cas9n is an efficient way to edit NK cells.The expression of IL-2 in NK cells are increased after CRISPR/Cas9 genome editing.