This invention discloses a gRNA combination and its application in the preparation of drugs for the prevention of metabolic-associated
fatty liver disease (MASLD). The gRNA combination, when mixed with Cas9
protein, yields an RNP complex, which, when microinjected into mouse zygotes, can
breed a stable and heritable
mouse strain with TMEM68
gene knockout. Combining lipidomics, transcriptomics, and primary
hepatocyte functional verification, the core regulatory role of TMEM68 in
hepatic lipid metabolism is revealed for the first time systematically. Experiments demonstrate that TMEM68 deficiency significantly reduces the storage of triglycerides (TAG) in the liver and hepatocytes, decreases
lipid droplet formation, and reshapes the metabolic
homeostasis of various lipids such as
glycerophospholipids,
cholesterol esters, and bile acids. Therefore, the gRNA combination of this invention, or the RNP complex obtained by mixing the gRNA combination with Cas9
protein, can be used to prepare drugs for the prevention of MASLD, providing a novel intervention strategy for MASLD prevention with broad application prospects.