RNA knockdown in vivo carries significant potential for disease modeling and therapies. Despite the emerging approaches of CRISPR/Cas9-mediated permanent knock out of targeted genes, strategies targeting RNA for disruption are advantageous in the treatment of acquired metabolic disorders when permanent modification of genome DNA is not appropriate, and RNA virus infection diseases when pathogenic DNA is not available (such as SARS-Cov-2 and MERS infections). Recently, Cas13d, a family of RNA-targeting CRISPR effectors, has been shown to accomplish robust down-regulation of cellular RNAs in mammalian cells in vitro (Konermann et al., 2018). Among the various Cas13d subtypes, CasRx (RfxCas13d) showed the most potent RNA knockdown efficiency in HEK293T cells (Konermann et al., 2018). However, the RNA-targeting activity of Cas13d still needed to be verified in vivo. In this study, the CasRx system was demonstrated to efficiently and functionally knock down genes related to metabolism functions, including Pten, Pcsk9 and lncLstr, in mouse hepatocytes. CasRx-mediated simultaneous knockdown of multiple genes was also achieved by sgRNA arrays, providing a useful strategy to modulate complex metabolism networks. Moreover, the AAV (adeno-associated virus)-mediated delivery of CasRx and Pcsk9 sgRNAs into mouse liver successfully decreased serum PCSK9, resulting in significant reduction of serum cholesterol levels. Importantly, CasRx-mediated knockdown of Pcsk9 is reversible and Pcsk9 could be repeatedly downregulated, providing an effective strategy to reversibly modulate metabolic genes. The present work supplies a successful proof-of-concept trial that suggests efficient and regulatory knockdown of target metabolic genes for a designed metabolism modulation in the liver. Targeted inhibition of a metabolism regulatory gene is often used for modeling and developing therapies of metabolic diseases (Moller, 2012). Recent years, many strategies of metabolic regulation were achieved using various modulators, including numerous small molecular compounds, nucleic acids, and therapeutic polypeptides/proteins targeting individual or multiple defined molecular products, such as enzymes, circulating proteins, cell-surface receptors and cellular RNAs (Moller, 2012). Applications of metabolic modulation have great potential for disease modeling and therapies. However, developing novel approaches with more specific and more flexible modulation of metabolic genes in vivo is still challenging, because natural and synthesized modulators with high targeting specificity are theoretically limited.

In recent years, an increasing number of genetic modification tools has emerged. Representing one of the greatest breakthroughs, the CRISPR/Cas system offers sequencespecific DNA editing methods to correct mutant genes in inherited metabolic diseases, and shows remarkable benefits to the establishment of metabolic disease animal models, such as inherited tyrosinemia (Rossidis et al., 2018). However, permanent modification of DNA is usually not an optimal strategy for the therapies of acquired metabolic disorders.