[YTHDF1 enhances the expression of exogenous proteins in the CHO-K1-GS^‒/‒ cell line]

YTH domain family protein 1 (YTHDF1) is an RNA-binding protein and belongs to the reader modified by N⁶-methyladenosine (m⁶A). It recognizes and binds to m⁶A modifications on RNA through specific domains, thereby performing corresponding biological functions. This study aims to explore the effects of YTHDF1 on the expression levels of exogenous proteins in the Chinese hamster ovary (CHO) cell line and evaluate its application potential in the optimization of the expression system of CHO cells. Firstly, the glutamine synthetase (GS) gene of CHO-K1 cells was knocked out by CRISPR/Cas9 to obtain the CHO-K1-GS^‒/‒ cell line. The enhancement effects of YTHDF1 overexpression on the expression of exogenous proteins in CHO-K1-GS^‒/‒ cells were further analyzed. Western blotting, qPCR, and fluorescence microscopy observations showed that the overexpression of YTHDF1 significantly up-regulated the expression levels of human serum albumin, single-chain antibody, and green fluorescent protein in cells. Moreover, this process did not have a significantly negative impact on the long-term proliferation and survival rate of the cells. In addition, the experiment with the translation inhibitor cycloheximide (CHX) confirmed that YTHDF1 mainly enhanced the protein expression by promoting translation in cells. This study demonstrates that YTHDF1 can enhance the cell's ability to synthesize exogenous proteins by promoting the translation process of mRNA. This strategy provides a new theoretical basis and technical direction for constructing efficient cell lines for producing biopharmaceuticals.