Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver.
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ABSTRACT:
SUBMITTER: He B
PROVIDER: S-EPMC7095259 | biostudies-literature | 2020 Jul
REPOSITORIES: biostudies-literature
Publications
Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver.
He Bingbing B Peng Wenbo W Huang Jia J Zhang Hang H Zhou Yingsi Y Yang Xiali X Liu Jing J Li Zhijie Z Xu Chunlong C Xue Mingxing M Yang Hui H Huang Pengyu P
Protein & cell 20200701 7
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Project description:RNA-guided and RNA-targeting type IV-D CRISPR/Cas systems (CRISPR/Cas13d) have recently been identified and employed for efficient and specific RNA knockdown in mammalian and plant cells. Cas13d possesses dual RNase activities and is capable of processing CRISPR arrays and cleaving target RNAs in a protospacer flanking sequence (PFS)-independent manner. These properties make this system a promising tool for multiplex gene expression regulation in microbes. Herein, we aimed to establish a CRISPR/Cas13d-mediated RNA knockdown platform for bacterial chassis. CasRx, Cas13d from Ruminococcus flavefaciens XPD3002, was selected due to its high activity. However, CasRx was found to be highly toxic to both Escherichia coli and Corynebacterium glutamicum, especially when it cooperated with its guide and target RNAs. After employing a low copy number vector, a tightly controlled promoter, and a weakened ribosome binding site, we successfully constructed an inducible expression system for CasRx and applied it for repressing the expression of a green fluorescent protein (GFP) in E. coli. Despite our efforts to optimize inducer usage, guide RNA (gRNA) architecture and combination, and target gene expression level, the highest gene repression efficiency was 30-50% at the protein level and ∼70% at the mRNA level. The moderate RNA knockdown is possibly caused by the collateral cleavage activity toward bystander RNAs, which acts as a mechanism of type IV-D immunity and perturbs microbial metabolism. Further studies on cellular response to CRISPR/Cas13d and improvement in RNA knockdown efficiency are required prior to practical application of this system in microbes.
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Project description:Type VI CRISPR effector Cas13d from Ruminococcus flavefaciens XPD3002 (RfxCas13d) is an RNA-guided RNA endonuclease. RfxCas13d has been harnessed to knockdown gene expression with high specificity in various systems including mammalian cells. While inducible knockdown is advantageous over constitutive knockdown in many scenarios, current inducible systems of RfxCas13d express CRISPR RNA and Cas13d separately. Such systems could be cumbersome to handle and may hamper the application of RfxCas13d in some scenarios. Here, we design an all-in-one Cas13d lentivirus vector which renders efficient and inducible knockdown in a doxycycline dosage-dependent manner. Furthermore, we find that Cas13d has a short half-life in mammalian cells. As a result, knockdown can be promptly reversed after doxycycline withdrawal. This vector is particularly useful for applications involving indispensable genes and/or in cells hard to transduce.
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Project description:An efficient mRNA knockdown strategy is needed to explore gene function in cells and embryos, especially to understand the process of maternal mRNA decay during early embryo development. Cas13, a novel RNA-targeting CRISPR effector protein, could bind and cleave complementary single-strand RNA, which has been employed for mRNA knockdown in mouse and human cells and RNA-virus interference in plants. Cas13 has not yet been reported to be used in pigs. In the current study, we explored the feasibility of CRISPR/Cas13d-mediated endogenous RNA knockdown in pigs. KDM5B, a histone demethylase of H3K4me3, was downregulated at the transcriptional level by 50% with CRISPR/Cas13d in porcine fibroblast cells. Knockdown of KDM5B-induced H3K4me3 expression and decreased the abundance of H3K27me3, H3K9me3, H3K4ac, H4K8ac, and H4K12ac. These changes affected cell proliferation and cell cycle. Furthermore, stable integration of the CRISPR/Cas13d system into the porcine genome resulted in the continuous expression of Cas13d and persistent knockdown of KDM5B. Finally, the RNA-targeting potential of Cas13d was further validated in porcine parthenogenetic embryos. By microinjection of Cas13d mRNA and gRNA targeting KDM5B into porcine oocytes, the expression of KDM5B was downregulated, the abundance of H3K4me3 increased as expected, and the expression of embryonic development-related genes was changed accordingly. These results indicate that CRISPR/Cas13d provides an easily programmable platform for spatiotemporal transcriptional manipulation in pigs.
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Project description:The chick embryo is a classical model system commonly used in developmental biology due to its amenability to gene perturbation experiments. Pairing this powerful model organism with cutting-edge technology can significantly expand the range of experiments that can be performed. Recently, the CRISPR-Cas13d system has been successfully adapted for use in zebrafish, medaka, killifish, and mouse embryos to achieve targeted gene expression knockdown. Despite its success in other animal models, no prior study has explored the potential of CRISPR-Cas13d in the chick. Here, we present an adaptation of the CRISPR-Cas13d system to achieve targeted gene expression knockdown in the chick embryo. As proof-of-principle, we demonstrate the knockdown of PAX7, an early neural crest marker. Application of this adapted CRISPR-Cas13d technique resulted in effective knockdown of PAX7 expression and function, comparable to knockdown achieved by translation-blocking morpholino. CRISPR-Cas13d complements preexisting knockdown tools such as CRISPR-Cas9 and morpholinos, thereby expanding the experimental potential and versatility of the chick model system.
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Project description:Even though insulin-like growth factor 2 (IGF2) has been reported to be overexpressed in nonalcoholic fatty liver disease (NAFLD), its role in the progression of NAFLD and the potential mechanism remain largely unclear. Using in vitro models, we found that IGF2 was the key overexpressed gene in steatosis, suggesting a possible association between IGF2 and NAFLD. Interestingly, loss-of-function experiments revealed that inhibition of IGF2 protein impaired mitochondrial biogenesis and respiration. It additionally disrupted the expression changes of mitochondrial fusion and fission-related proteins necessary in maintaining mitochondrial homeostasis. Consistently, IGF2 knockdown reduced the mitochondrial membrane potential and increased the production of reactive oxygen species. Mechanistically, IGF2 regulates mitochondrial functions by modulating the expression of SIRT1 and its downstream gene PGC1α. This research opens a new frontier on the role of IGF2 in energy metabolism, which potentially participates in the development of NAFLD. As such, IGF2 is a potential therapeutic target against NAFLD.
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Project description:To examine the potential off-target effects by CRISPR/Cas13d-mediated CTSL knockdown in 293FT cells
2022-06-09 | GSE203052 | GEO
Project description:BackgroundHistidine metabolism is crucial in role in tumor biology, contributing to tumor progression, immune regulation, and metabolic reprogramming. In hepatocellular carcinoma (HCC), dysregulated histidine metabolism may promote tumor growth and immune evasion, although the specific mechanisms remain poorly understood.MethodsUsing single-cell RNA sequencing, the expression patterns of histidine metabolism-related genes were evaluated across different cell types in HCC samples. In vivo and in vitro experiments were conducted to validate how histidine treatment affects macrophage and T-cell function. Furthermore, the TCGA database was utilized to construct a prognostic model to identify the key gene BUD23 and to examine its correlation with metabolism and immune infiltration.ResultsThe proportion of parenchymal cells exhibiting high histidine metabolism was significantly increased, accompanied by a general reduction in immune and stromal cell infiltration. Notably, macrophages and T cells demonstrated impaired antitumor functions. In the high histidine metabolism group, multiple critical cell communication pathways (e.g., MIF, CLEC, MHC II) were downregulated, macrophages shifted toward immunosuppressive subpopulations, T cells exhibited an exhaustion phenotype, and CD8 + T-cell activation was diminished. Further in vivo and in vitro co-culture experiments confirmed that elevated histidine concentrations promoted M2 polarization in macrophages and weakened T-cell cytotoxicity, accelerating tumor proliferation. According to TCGA analyses, BUD23 was upregulated in the high histidine metabolism group and significantly negatively correlated with patient survival and immune cell infiltration. Silencing BUD23 boosted immune cell activation and cytotoxic effects, effectively reversing the immunosuppressive microenvironment. A multivariable Cox regression-based prognostic model indicated unfavorable outcomes in patients with high histidine metabolism.ConclusionHistidine metabolism drives tumor cell metabolic reprogramming and reshapes the tumor immune microenvironment through intercellular communication, thereby promoting tumor progression. BUD23 shows promise as a biomarker for prognosis and immune response prediction in liver cancer. This study provides new therapeutic targets and theoretical support for liver cancer treatment by targeting histidine metabolism.
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Project description:SARS-CoV-2 entry into cells requires specific host proteases; however, no successful in vivo applications of host protease inhibitors have yet been reported for treatment of SARS-CoV-2 pathogenesis. Here we describe a chemically engineered nanosystem encapsulating CRISPR-Cas13d, developed to specifically target lung protease cathepsin L (Ctsl) messenger RNA to block SARS-CoV-2 infection in mice. We show that this nanosystem decreases lung Ctsl expression in normal mice efficiently, specifically and safely. We further show that this approach extends survival of mice lethally infected with SARS-CoV-2, correlating with decreased lung virus burden, reduced expression of proinflammatory cytokines/chemokines and diminished severity of pulmonary interstitial inflammation. Postinfection treatment by this nanosystem dramatically lowers the lung virus burden and alleviates virus-induced pathological changes. Our results indicate that targeting lung protease mRNA by Cas13d nanosystem represents a unique strategy for controlling SARS-CoV-2 infection and demonstrate that CRISPR can be used as a potential treatment for SARS-CoV-2 infection.
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Project description:PurposeCR increases fatty acid oxidation to decrease tissue lipid content. The Nuclear factor E2-related factor 2 (Nrf2)-Kelch like ECH associated Protein 1 (Keap1) pathway is an antioxidant gene regulatory pathway that has been previously investigated in weight gain. However, limited interaction of Nrf2/Keap1 and CR exists. The purpose of this study was to determine how Keap1 knockdown (Keap1-KD), which is known to increase Nrf2 activity, affects the CR response, such as weight loss, hepatic lipid decrease, and induction of fatty acid oxidation gene expression.MethodsC57BL/6 and Keap1-KD mice were maintained on 40% CR or fed ad libitum for 6 weeks. Hepatic lipid content, lipid metabolic gene, and miRNA expression was quantified.ResultsCR lowered hepatic lipid content, and induced fatty acid oxidation gene expression to a greater degree in Keap1-KD compared to C57BL/6 mice. CR differentially altered miRNA 34a, 370, let-7b* in livers of Keap1-KD compared to C57BL/6 mice.ConclusionsCR induced induction of fatty acid oxidation gene expression was augmented with Keap1 knockdown, which was associated with differential expression of several miRNAs implicated in fatty acid oxidation and lipid accumulation.
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