CRISPR-Cas9 Gene Research Articles

Generation of a new DiCre expressing parasite strain for functional characterization of Plasmodium falciparum genes in blood stages

Conditional regulation is a highly beneficial system for studying the function of essential genes in Plasmodium falciparum and dimerizable Cre recombinase (DiCre) is a recently adapted conditional regulation system suitable for this purpose. In the DiCre system, two inactive fragments of Cre are reconstituted to form a functionally active enzyme in the presence of rapamycin. Different loci have been targeted to generate parasite lines that express the DiCre enzyme. Here, we have used marker-free CRISPR-Cas9 gene editing to integrate the DiCre cassette in a redundant cg6 locus. We have shown the utility of the newly generated ∆cg6DC4 parasites in mediating robust, rapid, and highly specific excision of exogenously encoded gfp sequence. The ∆cg6DC4 parasites are also capable of conditional excision of an endogenous parasite gene, PF3D7_1246000. Conditional deletion of PF3D7_1246000 did not cause any inhibition in the asexual proliferation of the parasites. Furthermore, the health and morphology of the mutant parasites were comparable to that of the control parasites in Giemsa smears. The availability of another stable DiCre parasite strain competent for conditional excision of target genes will expedite functional characterization and validation of novel drug and vaccine targets against malaria.

Amino acid-level differences in alpha tubulin sequences are uniquely required for meiosis.

Tubulin is the major structural constituent of the microtubule cytoskeleton. Yeast Schizosaccharomyces pombe contain two α- tubulins genes, nda2 and atb2, that are highly functionally distinct: nda2 deletion is lethal, while lack of atb2 does not interfere with cell viability. The functional determinants underlying this distinction are unknown. Here we used CRISPR-Cas9 gene editing to generate a yeast strain expressing Atb2 amino acid sequence utilizing Nda2 codon usage in the native Nda2 locus. Such Nda2-coded Atb2 (NCA) yeast, unlike Nda2 knockout, were viable and displayed no visible abnormalities in cell morphology or vegetative life cycle. However, these NCA yeast showed strong impairments in sporulation and meiosis, including major meiotic delays and high rates of abnormal chromosome segregation. Our data indicate that the amino acid sequence of Nda2 is uniquely required for normal meiosis, and identify a novel determinant that underlies functional distinction between closely related tubulin isoforms.

Impaired fatty acid import or catabolism in macrophages restricts intracellular growth of Mycobacterium tuberculosis.

Mycobacterium tuberculosis ( Mtb ) infection of macrophages reprograms cellular metabolism to promote lipid retention. While it is clearly known that intracellular Mtb utilize host derived lipids to maintain infection, the role of macrophage lipid processing on the bacteria's ability to access the intracellular lipid pool remains undefined. We utilized a CRISPR-Cas9 genetic approach to assess the impact of sequential steps in fatty acid metabolism on the growth of intracellular Mtb . Our analyzes demonstrate that mutated macrophages that cannot either import, store or catabolize fatty acids restrict Mtb growth by both common and divergent anti-microbial mechanisms, including increased glycolysis, increased oxidative stress, production of pro-inflammatory cytokines, enhanced autophagy and nutrient limitation. We also show that impaired macrophage lipid droplet biogenesis is restrictive to Mtb replication, but increased induction fails to rescue Mtb growth. Our work expands our understanding of how host fatty acid homeostasis impacts Mtb growth in the macrophage.

Engineering Bacterial Chitinases for Industrial Application: From Protein Engineering to Bacterial Strains Mutation! A Comprehensive Review of Physical, Molecular, and Computational Approaches.

Bacterial chitinases are integral in breaking down chitin, the natural polymer in crustacean and insect exoskeletons. Their increasing utilization across various sectors such as agriculture, waste management, biotechnology, food processing, and pharmaceutical industries highlights their significance as biocatalysts. The current review investigates various scientific strategies to maximize the efficiency and production of bacterial chitinases for industrial use. Our goal is to optimize the heterologous production process using physical, molecular, and computational tools. Physical methods focus on isolating, purifying, and characterizing chitinases from various sources to ensure optimal conditions for maximum enzyme activity. Molecular techniques involve gene cloning, site-directed mutation, and CRISPR-Cas9 gene editing as an approach for creating chitinases with improved catalytic activity, substrate specificity, and stability. Computational approaches use molecular modeling, docking, and simulation techniques to accurately predict enzyme-substrate interactions and enhance chitinase variants' design. Integrating multidisciplinary strategies enables the development of highly efficient chitinases tailored for specific industrial applications. This review summarizes current knowledge and advances in chitinase engineering to serve as an indispensable guideline for researchers and industrialists seeking to optimize chitinase production for various uses.

ARF1 compartments direct cargo flow via maturation into recycling endosomes.

Cellular membrane homoeostasis is maintained via a tightly regulated membrane and cargo flow between organelles of the endocytic and secretory pathways. Adaptor protein complexes (APs), which are recruited to membranes by the small GTPase ARF1, facilitate cargo selection and incorporation into trafficking intermediates. According to the classical model, small vesicles would facilitate bi-directional long-range transport between the Golgi, endosomes and plasma membrane. Here we revisit the intracellular organization of the vesicular transport machinery using a combination of CRISPR-Cas9 gene editing, live-cell high temporal (fast confocal) or spatial (stimulated emission depletion) microscopy as well as correlative light and electron microscopy. We characterize tubulo-vesicular ARF1 compartments that harbour clathrin and different APs. Our findings reveal two functionally different classes of ARF1 compartments, each decorated by a different combination of APs. Perinuclear ARF1 compartments facilitate Golgi export of secretory cargo, while peripheral ARF1 compartments are involved in endocytic recycling downstream of early endosomes. Contrary to the classical model of long-range vesicle shuttling, we observe that ARF1 compartments shed ARF1 and mature into recycling endosomes. This maturation process is impaired in the absence of AP-1 and results in trafficking defects. Collectively, these data highlight a crucial role for ARF1 compartments in post-Golgi sorting.

High TNF and NF-ĸB pathway dependency are associated with AZD5582 sensitivity in OSCC via CASP8-dependent apoptosis.

Drug repurposing can accelerate the development of novel therapeutic strategies. Oral squamous cell carcinoma (OSCC) is prevalent in Asians and is associated with a poor prognosis. Here, we integrate newly generated sensitivity data for 339 anticancer drugs with genome-wide CRISPR‒Cas9 gene essentiality data for 21 cell lines mostly derived from Asian OSCC patients to identify drug repurposing candidates. We observe potent activity of AZD5582 (an antagonist of the inhibitor of apoptosis (IAP) family of proteins) in a subset of OSCC cells, which was associated with dependency on NF-ĸB pathway genes (RNF31, MAP3K7 and IKBKG). We confirm the on-target specificity and potency of AZD5582 in vitro and its ability to suppress cell growth in xenograft models. Furthermore, we identify tumor necrosis factor (TNF) as a key mediator of AZD5582 sensitivity in OSCC. CASP8-dependent apoptotic and CASP8-independent necroptotic cellular programs mediate AZD5582-induced cell death. In summary, through the systematic integration of pharmacological and CRISPR data, we identified a subset of OSCC with potent sensitivity to AZD5582 mediated through the NF-κB and TNF signalling pathways.

RNA lipid nanoparticles as efficient in vivo CRISPR-Cas9 gene editing tool for therapeutic target validation in glioblastoma cancer stem cells

In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ∼80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research.

The RNA helicase LOS4 regulates pre-mRNA splicing of key genes (EIN2, ERS2, CTR1) in the ethylene signaling pathway.

The Arabidopsis RNA helicase LOS4 plays a key role in regulating pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. The plant hormone ethylene plays diverse roles in plant growth, development, and responses to stress. Ethylene is perceived by the membrane-bound ethylene receptors complex, and then triggers downstream components, such as EIN2, to initiate signal transduction into the nucleus, leading to the activation of ethylene-responsive genes. Over the past decades, substantial information has been accumulated regarding gene cloning, protein-protein interactions, and downstream gene expressions in the ethylene pathway. However, our understanding of mRNA post-transcriptional processing and modification of key genes in the ethylene signaling pathway remains limited. This study aims to provide evidence demonstrating the involvement of the Arabidopsis RNA helicase LOS4 in pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. Various genetic approaches including RNAi gene silencing, CRISPR-Cas9 gene editing, and amino acid mutations were employed in this study. When LOS4 was silenced or knocked down, the ethylene sensitivity of etiolated seedlings was significantly enhanced. Further investigation revealed errors in the EIN2 pre-mRNA splicing when LOS4 was knocked down. In addition, aberrant pre-mRNA splicing was observed in the ERS2 and CTR1 genes in the pathway. Biochemical assays indicated that the los4-2 (E94K) mutant protein exhibited increased ATP binding and enhanced ATP hydrolytic activity. Conversely, the los4-1 (G364R) mutant had reduced substrate RNA binding and lower ATP binding activities. These findings significantly advanced our comprehension of the regulatory functions and molecular mechanisms of RNA helicase in ethylene signaling.

Reducing CRISPR-Cas9 off-target effects by optically controlled chemical modifications of guide RNA

A photocatalytic click chemistry approach, offering a significant advancement over conventional methods inRNA function modulation is described. This innovative method, utilizing light-activated small molecules, provides a high level of precision and control in RNA regulation, particularly effective in intricate cellular processes. By applying this strategy to CRISPR-Cas9 gene editing, we demonstrate its effectiveness in enhancing gene editing specificity and markedly reducing off-target effects. Our approach employs a vinyl ether modification in RNA, which activated under visible light with a phenanthrenequinone derivative, creating a CRISPR-OFF switch that precisely regulates CRISPR system activity. This method not only represents an advancement in genomic interventions but also offers broad applications in gene regulation, paving the way for safer and more reliable gene editing in therapeutic genomics.

The JAK inhibitor, Tofacitinib, Corrects the Overexpression of CEACAM6 and Limits Susceptibility to AIEC Caused by Reduced Activity of the IBD Associated Gene, PTPN2.

A cohort of patients with inflammatory bowel disease (IBD) exhibit expansion of the gut pathobiont, adherent-invasive E. coli (AIEC). Loss of activity of the IBD susceptibility gene, protein tyrosine phosphatase type 2 ( PTPN2 ), results in dysbiosis of the gut microbiota both in human subjects and mice. Further, constitutive Ptpn2 knock-out ( Ptpn2 -KO) mice display expansion of AIEC compared to wildtype littermates. CEACAM6, a host cell surface glycoprotein, is exploited by AIEC to attach to and enter intestinal epithelial cells (IECs). Here, we investigate the role of IEC-specific PTPN2 in restricting AIEC invasion. Biopsies from IBD patients heterozygous (CT) or homozygous (CC) for the PTPN2 SNP (single nucleotide polymorphism) rs1893217 were processed for immunohistochemistry. HT-29 intestinal epithelial cells (IEC) were transfected with control shRNA ( PTPN2 -CTL), or a shRNA targeted towards PTPN2 ( PTPN2 -KD). The rs1893217 SNP was inserted ( PTPN2 -KI), or a complete knock-out of PTPN2 ( PTPN2 - KO) was generated, with CRISPR-Cas9 gene editing of Caco-2BBe IEC lines. Adherence and invasion assays were performed with either the human IBD AIEC isolate, LF82, or a novel fluorescent-tagged mouse adherent-invasive E. coli ( m AIEC red ) at multiplicity of infection (MOI) of 10. IL-6 and the pan-JAK inhibitor tofacitinib were administered to interrogate JAK-STAT signaling. Protein expression was determined by western blotting and densitometry. CEACAM6 expression was elevated (colon and ileum) in IBD patients carrying the PTPN2 rs1893217 SNP (CT, CC) compared to wildtype (TT) IBD patients. HT-29 and Caco-2BBe cell lines deficient in PTPN2 expressed significantly higher levels of CEACAM6. Further, PTPN2 -KI and PTPN2 -KO cell lines also displayed greater adherence and invasion by AIEC LF82 and higher m AIEC red invasion. CEACAM6 expression was further elevated after administration of IL-6 in PTPN2 -deficient cell lines compared to untreated controls. Silencing of STAT1 and 3 partially reduced CEACAM6 protein expression. Tofacitinib significantly reduced the elevated CEACAM6 protein expression and the higher AIEC adherence and invasion in PTPN2 -KI and PTPN2-KO cell lines compared to DMSO controls. Our findings highlight a crucial role for PTPN2 in restricting pathobiont entry into host cells. Our study also describes a role for the FDA-approved drug, tofacitinib (Xeljanz) in correcting the JAK-STAT- mediated over-expression of CEACAM6, used by pathobionts as an entry portal into host cells. These findings suggest a role for JAK-inhibitors in mitigating AIEC colonization in IBD-susceptible hosts.

Integrating Artificial Intelligence and Advanced Genomic Technologies in Unraveling Autism Spectrum Disorder and Gastrointestinal Comorbidities: A Multidisciplinary Approach to Precision Medicine

This article explores the potential impact of Artificial Intelligence (AI), Machine Learning (ML), CRISPR-Cas9 gene editing, and single-cell RNA sequencing on improving our understanding and management of Autism Spectrum Disorder (ASD) and its gastrointestinal (GI) comorbidities. It examines how these technologies illuminate the complex interplay between the gut and the brain, identifying specific enzyme deficiencies and microbial imbalances linked to GI symptoms in ASD. By leveraging AI and ML, personalized intervention strategies are developed through the analysis of genomic, proteomic, and environmental data, enhancing our ability to predict and address GI issues in ASD. Additionally, CRISPR-Cas9 gene editing holds promise for correcting genetic abnormalities related to enzyme production, potentially offering precise treatments. Single-cell RNA sequencing provides critical insights into the cellular diversity of the ASD gut, uncovering new therapeutic targets. The article highlights the transformative potential of these technologies while addressing the associated challenges and ethical considerations. It underscores the necessity of a multidisciplinary approach to fully harness their benefits and discusses the significant progress and emerging trends in the field, emphasizing the role of technological advancements in advancing precision medicine for ASD and its GI comorbidities.

Developmental interplay between transcriptional alterations and a targetable cytokine signaling dependency in pediatric ETO2::GLIS2 leukemia

BackgroundSeveral fusion oncogenes showing a higher incidence in pediatric acute myeloid leukemia (AML) are associated with heterogeneous megakaryoblastic and other myeloid features. Here we addressed how developmental mechanisms influence human leukemogenesis by ETO2::GLIS2, associated with dismal prognosis.MethodsWe created novel ETO2::GLIS2 models of leukemogenesis through lentiviral transduction and CRISPR-Cas9 gene editing of human fetal and post-natal hematopoietic stem/progenitor cells (HSPCs), performed in-depth characterization of ETO2::GLIS2 transformed cells through multiple omics and compared them to patient samples. This led to a preclinical assay using patient-derived-xenograft models to test a combination of two clinically-relevant molecules.ResultsWe showed that ETO2::GLIS2 expression in primary human fetal CD34+ hematopoietic cells led to more efficient in vivo leukemia development than expression in post-natal cells. Moreover, cord blood-derived leukemogenesis has a major dependency on the presence of human cytokines, including IL3 and SCF. Single cell transcriptomes revealed that this cytokine environment controlled two ETO2::GLIS2-transformed states that were also observed in primary patient cells. Importantly, this cytokine sensitivity may be therapeutically-exploited as combined MEK and BCL2 inhibition showed higher efficiency than individual molecules to reduce leukemia progression in vivo.ConclusionsOur study uncovers an interplay between the cytokine milieu and transcriptional programs that extends a developmental window of permissiveness to transformation by the ETO2::GLIS2 AML fusion oncogene, controls the intratumoral cellular heterogeneity, and offers a ground-breaking therapeutical opportunity by a targeted combination strategy.

Kinesin-7 CENP-E mediates centrosome organization and spindle assembly to regulate chromosome alignment and genome stability.

Chromosome congression and alignment are essential for cell cycle progression and genomic stability. Kinesin-7 CENP-E, a plus-end-directed kinesin motor, is required for chromosome biorientation, congression and alignment in cell division. However, it remains unclear how chromosomes are aligned and segregated in the absence of CENP-E in mitosis. In this study, we utilize the CRISPR-Cas9 gene editing method and high-throughput screening to establish CENP-E knockout cell lines and reveal that CENP-E deletion results in defects in chromosome congression, alignment and segregation, which further promotes aneuploidy and genomic instability in mitosis. Both CENP-E inhibition and deletion lead to the dispersion of spindle poles, the formation of the multipolar spindle and spindle disorganization, which indicates that CENP-E is necessary for the organization and maintenance of spindle poles. In addition, CENP-E heterozygous deletion in spleen tissues also leads to the accumulation of dividing lymphocytes and cell cycle arrest in vivo. Furthermore, CENP-E deletion also disrupts the localization of key kinetochore proteins and triggers the activation of the spindle assembly checkpoint. In summary, our findings demonstrate that CENP-E promotes kinetochore-microtubule attachment and spindle pole organization to regulate chromosome alignment and spindle assembly checkpoint during cell division.

PTPRJ is a negative regulator of insulin signaling in neuronal cells, impacting protein biosynthesis, and neurite outgrowth.

Central insulin resistance has been linked to the development of neurodegenerative diseases and mood disorders. Various proteins belonging to the enzyme family of protein tyrosine phosphatases (PTPs) act as inhibitors of insulin signaling. Protein tyrosine phosphatase receptor type J (PTPRJ) has been identified as a negative regulator in insulin signaling in the periphery. However, the impact of PTPRJ on insulin signaling and its functional role in neuronal cells is largely unknown. Therefore, we generated a Ptprj knockout (KO) cell model in the murine neuroblast cell line Neuro2a by CRISPR-Cas9 gene editing. Ptprj KO cells displayed enhanced insulin signaling, as shown by increased phosphorylation of the insulin receptor (INSR), IRS-1, AKT, and ERK1/2. Further, proximity ligation assays (PLA) revealed both direct interaction of PTPRJ with the INSR and recruitment of this phosphatase to the receptor upon insulin stimulation. By RNA sequencing gene expression analysis, we identified multiple gene clusters responsible for glucose uptake and metabolism, and genes involved in the synthesis of various lipids being mainly upregulated under PTPRJ deficiency. Furthermore, multiple Ca2+ transporters were differentially expressed along with decreased protein biosynthesis. This was accompanied by an increase in endoplasmic reticulum (ER) stress markers. On a functional level, PTPRJ deficiency compromised cell differentiation and neurite outgrowth, suggesting a role in nervous system development. Taken together, PTPRJ emerges as a negative regulator of central insulin signaling, impacting neuronal metabolism and neurite outgrowth.

Heterogeneity in establishment of polyethylene glycol-mediated plasmid transformations for five forest pathogenic Phytophthora species.

Plasmid-mediated DNA transformation is a foundational molecular technique and the basis for most CRISPR-Cas9 gene editing systems. While plasmid transformations are well established for many agricultural Phytophthora pathogens, development of this technique in forest Phytophthoras is lacking. Given our long-term research objective to develop CRISPR-Cas9 gene editing in a forest pathogenic Phytophthora species, we sought to establish the functionality of polyethylene glycol (PEG)-mediated plasmid transformation in five species: P. cactorum, P. cinnamomi, P. cryptogea, P. ramorum, and P. syringae. We used the agricultural pathogen P. sojae, a species for which PEG-mediated transformations are well-established, as a transformation control. Using a protocol previously optimized for P. sojae, we tested transformations in the five forest Phytophthoras with three different plasmids: two developed for CRISPR-Cas9 gene editing and one developed for fluorescent protein tagging. Out of the five species tested, successful transformation, as indicated by stable growth of transformants on a high concentration of antibiotic selective growth medium and diagnostic PCR, was achieved only with P. cactorum and P. ramorum. However, while transformations in P. cactorum were consistent and stable, transformations in P. ramorum were highly variable and yielded transformants with very weak mycelial growth and abnormal morphology. Our results indicate that P. cactorum is the best candidate to move forward with CRISPR-Cas9 protocol development and provide insight for future optimization of plasmid transformations in forest Phytophthoras.

Step-wise approach for In silico CRISPR-Cas9 gene editing of IVS I-110 and IVS I-6 Beta-thalassemia-causing variants

Step-wise approach for In silico CRISPR-Cas9 gene editing of IVS I-110 and IVS I-6 Beta-thalassemia-causing variants

Polymer-locking fusogenic liposomes for glioblastoma-targeted siRNA delivery and CRISPR-Cas gene editing.

In patients with glioblastoma (GBM), upregulated midkine (MDK) limits the survival benefits conferred by temozolomide (TMZ). RNA interference (RNAi) and CRISPR-Cas9 gene editing technology are attractive approaches for regulating MDK expression. However, delivering these biologics to GBM tissue is challenging. Here we demonstrate a polymer-locking fusogenic liposome (Plofsome) that can be transported across the blood-brain barrier (BBB) and deliver short interfering RNA or CRISPR-Cas9 ribonucleoprotein complexes into the cytoplasm of GBM cells. Plofsome is designed by integrating a 'lock' into the fusogenic liposome using a traceless reactive oxygen species (ROS)-cleavable linker so that fusion occurs only after crossing the BBB and entering the GBM tissue with high ROS levels. Our results showed that MDK suppression by Plofsomes significantly reduced TMZ resistance and inhibited GBM growth in orthotopic brain tumour models. Importantly, Plofsomes are effective only at tumour sites and not in normal tissues, which improves the safety of combined RNAi and CRISPR-Cas9 therapeutics.

Chemokine-mediated F-actin dynamics, polarity, and migration in B lymphocytes depend on WNK1 signaling.

Ligand-engaged chemokine receptors trigger nucleotide exchange in heterotrimeric Gαi proteins, which stimulates cytoskeletal reorganization and cell polarity changes. To better understand the signaling events responsible for these cellular changes, we focused on early changes in F-actin dynamics after engagement of the chemokine receptor CXCR5 in murine splenic B cells. Within 10 seconds of exposure to the CXCR5 ligand CXCL13, three-dimensional lamellar-like pseudopods and F-actin-rich ridges appeared. The transient F-actin increase depended on Gαi2/3 signaling, the PI3K/AKT pathway, ERK activation, phospholipase C activity, and Rac1/2 activation mediated by Dock2 (dedicator of cytokinesis 2). Immunoblot analyses identified the kinase WNK1 (with no lysine kinase 1) as a potential early AKT effector. Treating B cells with specific WNK inhibitors disrupted F-actin dynamics and impaired B cell polarity, motility, and chemotaxis. These changes were mimicked in a murine B cell line by CRISPR-Cas9 gene editing of Wnk1, which also suggested that WNK1 contributed to B cell proliferation. Administration of a single dose of a WNK inhibitor transiently reduced B cell motility and polarity in the lymph nodes of live mice. These results indicate that WNK1 signaling maintains B cell responsiveness to CXCL13 and suggest that pharmacological inhibition of WNK1, which is involved in cancer progression and blood pressure regulation, may affect humoral immunity.

TFEB activator tanshinone IIA and derivatives derived from Salvia miltiorrhiza Bge. Attenuate hepatic steatosis and insulin resistance

Ethnopharmacological relevanceSalvia miltiorrhiza Bge. (SMB) is an herbal medicine extensively used for improving metabolic disorders, including Nonalcoholic fatty liver disease (NAFLD). However, the potential material basis and working mechanism still remained to be elucidated. Aim of the studyTo find potential ingredients for therapy of NAFLD by high content screening and further verify the efficacy on restoring hepatic steatosis and insulin resistance, and clarify the potential working mechanism. Materials and methodsThe mouse transcription factor EB (Tfeb) in preadipocytes was knocked out by CRISPR-Cas9 gene editing. High content screening of TFEB nuclear translocation was performed to identify TFEB activators. The effect of candidate compounds on reducing lipid accumulation was evaluated using Caenorhabditis elegans (C. elegans). Then the role of Salvia miltiorrhiza extract (SMB) containing Tanshinone IIA and the derivatives were further investigated on high-fat diet (HFD) fed mice. RNA-seq was performed to explore potential molecular mechanism of SMB. Finally, the gut microbiota diversity was evaluated using 16S rRNA sequencing to investigate the protective role of SMB on regulating gut microbiota homeostasis. ResultsKnockout of Tfeb led to excessive lipid accumulation in adipocytes while expression of TFEB homolog HLH-30 in C. elegans (MAH240) attenuated lipid deposition. Screening of TFEB activators identified multiple candidates from Salvia miltiorrhiza, all of them markedly induced lysosome biogenesis in HepG2 cells. One of the candidate compounds Tanshinone IIA significantly decreased lipid droplet deposition in HFD fed C. elegans. Administration of SMB on C57BL/6J mice via gastric irrigation at the dose of 15 g/kg/d markedly alleviated hepatic steatosis, restored serum lipid profile, and glucose tolerance. RNA-seq showed that gene expression profile was altered and the genes related to lipid metabolism were restored. The disordered microbiome was remodeled by SMB, Firmicutes and Actinobacteriotawere notably reduced, Bacteroidota and Verrucomicrobiota were significantly increased. ConclusionTaken together, the observations presented here help address the question concerning what were the main active ingredients in SMB for alleviating NAFLD, and established that targeting TFEB was key molecular basis for the efficacy of SMB.

Functional analysis of epilepsy-associated GABAA receptor mutations using Caenorhabditis elegans.

GABAA receptor subunit mutations pose a significant risk for genetic generalized epilepsy; however, there are over 150 identified variants, many with unknown or unvalidated pathogenicity. We aimed to develop invivo models for testing GABAA receptor variants using the model organism, Caenorhabditis elegans. CRISPR-Cas9 gene editing was used to create a complete deletion of unc-49, a C. elegans GABAA receptor, and to create homozygous epilepsy-associated mutations in the endogenous unc-49 gene. The unc-49 deletion strain was rescued with transgenes for either the C. elegans unc-49B subunit or the α1, β3, and γ2 subunits for the human GABAA receptor. All newly created strains were analyzed for phenotype and compared against existing unc-49 mutations. Nematodes with a full genetic deletion of the entire unc-49 locus were compared with existing unc-49 mutations in three separate phenotypic assays-coordinated locomotion, shrinker frequency and seizure-like convulsions. The full unc-49 deletion exhibited reduced locomotion and increased shrinker frequency and PTZ-induced convulsions, but were not found to be phenotypically stronger than existing unc-49 mutations. Rescue with the unc-49B subunit or creation of humanized worms for the GABAA receptor both showed partial phenotypic rescue for all three phenotypes investigated. Finally, two epilepsy-associated variants were analyzed and deemed to be loss of function, thus validating their pathogenicity. These findings establish C. elegans as a genetic model to investigate GABAA receptor mutations and delineate a platform for validating associated variants in any epilepsy-associated gene. Epilepsy is a complex human disease that can be caused by mutations in specific genes. Many possible mutations have been identified, but it is unknown for most of them whether they cause the disease. We tested the role of mutations in one specific gene using a small microscopic worm as an animal model. Our results establish this worm as a model for epilepsy and confirm that the two unknown mutations are likely to cause the disease.