The Yeast Permease Agp2 Senses Cycloheximide and Undergoes Degradation That Requires the Small Protein Brp1-Cellular Fate of Agp2 in Response to Cycloheximide.

The Saccharomyces cerevisiae Agp2 is a plasma membrane protein initially reported to be an uptake transporter for L-carnitine. Agp2 was later rediscovered, together with three additional proteins, Sky1, Ptk2, and Brp1, to be involved in the uptake of the polyamine analogue bleomycin-A5, an anticancer drug. Mutants lacking either Agp2, Sky1, Ptk2, or Brp1 are extremely resistant to polyamines and bleomycin-A5, suggesting that these four proteins act in the same transport pathway. We previously demonstrated that pretreating cells with the protein synthesis inhibitor cycloheximide (CHX) blocked the uptake of fluorescently labelled bleomycin (F-BLM), raising the possibility that CHX could either compete for F-BLM uptake or alter the transport function of Agp2. Herein, we showed that the agp2Δ mutant displayed striking resistance to CHX as compared to the parent, suggesting that Agp2 is required to mediate the physiological effect of CHX. We examined the fate of Agp2 as a GFP tag protein in response to CHX and observed that the drug triggered the disappearance of Agp2 in a concentration- and time-dependent manner. Immunoprecipitation analysis revealed that Agp2-GFP exists in higher molecular weight forms that were ubiquitinylated, which rapidly disappeared within 10 min of treatment with CHX. CHX did not trigger any significant loss of Agp2-GFP in the absence of the Brp1 protein; however, the role of Brp1 in this process remains elusive. We propose that Agp2 is degraded upon sensing CHX to downregulate further uptake of the drug and discuss the potential function of Brp1 in the degradation process.

Can Cisplatin Therapy Be Improved? Pathways That Can Be Targeted.

Cisplatin (cis-diamminedichloroplatinum (II)) is the oldest known chemotherapeutic agent. Since the identification of its anti-tumour activity, it earned a remarkable place as a treatment of choice for several cancer types. It remains effective against testicular, bladder, lung, head and neck, ovarian, and other cancers. Cisplatin treatment triggers different cellular responses. However, it exerts its cytotoxic effects by generating inter-strand and intra-strand crosslinks in DNA. Tumour cells often develop tolerance mechanisms by effectively repairing cisplatin-induced DNA lesions or tolerate the damage by adopting translesion DNA synthesis. Cisplatin-associated nephrotoxicity is also a huge challenge for effective therapy. Several preclinical and clinical studies attempted to understand the major limitations associated with cisplatin therapy, and so far, there is no definitive solution. As such, a more comprehensive molecular and genetic profiling of patients is needed to identify those individuals that can benefit from platinum therapy. Additionally, the treatment regimen can be improved by combining cisplatin with certain molecular targeted therapies to achieve a balance between tumour toxicity and tolerance mechanisms. In this review, we discuss the importance of various biological processes that contribute to the resistance of cisplatin and its derivatives. We aim to highlight the processes that can be modulated to suppress cisplatin resistance and provide an insight into the role of uptake transporters in enhancing drug efficacy.

Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis. In this review, we discuss the most recent efforts towards novel non-viral delivery systems, focusing on strategies and mechanisms of peptide-based delivery systems, that can specifically deliver CRISPR components to different cell types for therapeutic and research purposes.

RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors.

Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3::uidA targets in plant cells. Further, the dCas9:SRDX-mediated transcriptional repression of an endogenous gene. Thus, our results suggest that the synthetic transcriptional repressor (dCas9:SRDX) and activators (dCas9:EDLL and dCas9:TAD) can be used as endogenous transcription factors to repress or activate transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9 DNA-targeting platform can be used in plants as a functional genomics tool and for biotechnological applications.

Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae.

The development of highly efficient genome engineering reagents is of paramount importance to launch the next wave of biotechnology. TAL effectors have been developed as an adaptable DNA binding scaffold that can be engineered to bind to any user-defined sequence. Thus, TAL-based DNA binding modules have been used to generate chimeric proteins for a variety of targeted genome modifications across eukaryotic species. For example, TAL effectors fused to the catalytic domain of FokI endonuclease (TALENs) were used to generate site-specific double strand breaks (DSBs), the repair of which can be harnessed to dictate user-desired, genome-editing outcomes. To cleave DNA, FokI endonuclease must dimerize which can be achieved using a pair of TALENs that bind to the DNA targeted in a tail-to-tail orientation with proper spacing allowing the dimer formation. Because TALENs binding to DNA are dependent on their repeat sequences and nucleotides binding specificities, homodimers and heterodimers binding can be formed. In the present study, we used several TALEN monomers with increased repeats binding degeneracy to allow homodimer formation at increased number of genomic loci. We assessed their binding specificities and genome modification activities. Our results indicate that homodimeric TALENs could be used to modify the yeast genome in a site-specific manner and their binding to the promoter regions might modulate the expression of target genes. Taken together, our data indicate that homodimeric TALENs could be used to achieve different engineering possibilities of biotechnological applications and that their transcriptional modulations need to be considered when analyzing their phenotypic effects.

CRISPR-Based Multiplex Detection of Human Papillomaviruses for One-Pot Point-of-Care Diagnostics.

The World Health Organization's global initiative toward eliminating high-risk Human Papillomavirus (hrHPV)-related cancers recommends DNA testing over visual inspection in all settings for primary cancer screening and HPV eradication by 2100. However, multiple hrHPV types cause different types of cancers, and there is a pressing need for an easy-to-use, multiplex point-of-care diagnostic platform for detecting different hrHPV types. Recently, CRISPR-Cas systems have been repurposed for point-of-care detection. Here, we established a CRISPR-Cas multiplexed diagnostic assay (CRISPRD) to detect cervical cancer-causing hrHPVs in one reaction (one-pot assay). We harnessed the compatibility of thermostable AapCas12b, TccCas13a, and HheCas13a nucleases with isothermal amplification and successfully detected HPV16 and HPV18, along with an internal control in a single-pot assay with a limit of detection of 10 copies and 100% specificity. This platform offers a rapid and practical solution for the multiplex detection of hrHPVs, which may facilitate large-scale hrHPV point-of-care screening. Furthermore, the CRISPRD platform programmability enables it to be adapted for the multiplex detection of any two nucleic acid biomarkers as well as internal control.

Loss of the yeast transporter Agp2 upregulates the pleiotropic drug-resistant pump Pdr5 and confers resistance to the protein synthesis inhibitor cycloheximide.

The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5. Cells lacking Agp2 are hyper-resistant to polyamine and bleomycin-A5. In these earlier studies, we showed that the protein synthesis inhibitor cycloheximide blocked the uptake of bleomycin-A5 into the cells suggesting that the drug uptake system may require de novo synthesis. However, our recent findings demonstrated that cycloheximide, instead, induced rapid degradation of Agp2, and in the absence of Agp2 cells are resistant to cycloheximide. These observations raised the possibility that the degradation of Agp2 may allow the cell to alter its drug resistance network to combat the toxic effects of cycloheximide. In this study, we show that membrane extracts from agp2Δ mutants accentuated several proteins that were differentially expressed in comparison to the parent. Mass spectrometry analysis of the membrane extracts uncovered the pleiotropic drug efflux pump, Pdr5, involved in the efflux of cycloheximide, as a key protein upregulated in the agp2Δ mutant. Moreover, a global gene expression analysis revealed that 322 genes were differentially affected in the agp2Δ mutant versus the parent, including the prominent PDR5 gene and genes required for mitochondrial function. We further show that Agp2 is associated with the upstream region of the PDR5 gene, leading to the hypothesis that cycloheximide resistance displayed by the agp2Δ mutant is due to the derepression of the PDR5 gene.

A CRISPR-based approach using dead Cas9-sgRNA to detect SARS-CoV-2.

Rapid, highly specific, and robust diagnostic kits to detect viruses and pathogens are needed to control disease spread and transmission globally. Of the many different methods proposed to diagnose COVID-19 infection, CRISPR-based detection of nucleic acids tests are among the most prominent. Here, we describe a new way of using CRISPR/Cas systems as a rapid and highly specific tool to detect the SARS-CoV-2 virus using the in vitro dCas9-sgRNA-based technique. As a proof of concept, we used a synthetic DNA of the M gene, one of the SARS-CoV-2 virus genes, and demonstrated that we can specifically inactivate unique restriction enzyme sites on this gene using CRISPR/Cas multiplexing of dCas9-sgRNA-BbsI and dCas9-sgRNA-XbaI. These complexes recognize and bind to the target sequence spanning the BbsI and XbaI restriction enzyme sites, respectively, and protect the M gene from digestion by BbsI and/or XbaI. We further demonstrated that this approach can be used to detect the M gene when expressed in human cells and from individuals infected with SARS-CoV-2. We refer to this approach as dead Cas9 Protects Restriction Enzyme Sites, and believe that it has the potential to be applied as a diagnostic tool for many DNA/RNA pathogens.

Mft1, identified from a genome-wide screen of the yeast haploid mutants, mediates cell cycle arrest to counteract quinoxaline-induced toxicity.

Quinoxaline is a heterocyclic compound with a two-membered ring structure that undergoes redox cycling to produce toxic free radicals. It has antiviral, antibacterial, antifungal, and antitumor activities. However, the biological functions that are involved in mounting a response against the toxic effects of quinoxaline have not been investigated. Herein, we performed a genome-wide screen using the yeast haploid mutant collection and reported the identification of 12 mutants that displayed varying sensitivity towards quinoxaline. No mutant was recovered that showed resistance to quinoxaline. The quinoxaline-sensitive mutants were deleted for genes that encode cell cycle function, as well as genes that belong to other physiological pathways such as the vacuolar detoxification process. Three of the highly sensitive gene-deletion mutants lack the DDC1, DUN1, and MFT1 genes. While Ddc1 and Dun1 are known to perform roles in the cell cycle arrest pathway, the role of Mft1 remains unclear. We show that the mft1Δ mutant is as sensitive to quinoxaline as the ddc1Δ mutant. However, the double mutant ddc1Δ mft1Δ lacking the DDC1 and MFT1 genes, is extremely sensitive to quinoxaline, as compared to the ddc1Δ and mft1Δ single mutants. We further show that the mft1Δ mutant is unable to arrest in the G2/M phase in response to the drug. We conclude that Mft1 performs a unique function independent of Ddc1 in the cell cycle arrest pathway in response to quinoxaline exposure. This is the first demonstration that quinoxaline exerts its toxic effect likely by inducing oxidative DNA damage causing cell cycle arrest. We suggest that clinical applications of quinoxaline and its derivatives should entail targeting cancer cells with defective cell cycle arrest.

Altered Regulation of the Glucose Transporter GLUT3 in PRDX1 Null Cells Caused Hypersensitivity to Arsenite.

Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy.

Functional analysis of OsPUT1, a rice polyamine uptake transporter.

Polyamines are nitrogenous compounds found in all eukaryotic and prokaryotic cells and absolutely essential for cell viability. In plants, they regulate several growth and developmental processes and the levels of polyamines are also correlated with the plant responses to various biotic and abiotic stresses. In plant cells, polyamines are synthesized in plastids and cytosol. This biosynthetic compartmentation indicates that the specific transporters are essential to transport polyamines between the cellular compartments. In the present study, a phylogenetic analysis was used to identify candidate polyamine transporters in rice. A full-length cDNA rice clone AK068055 was heterologously expressed in the Saccharomyces cerevisiae spermidine uptake mutant, agp2∆. Radiological uptake and competitive inhibition studies with putrescine indicated that rice gene encodes a protein that functioned as a spermidine-preferential transporter. In competition experiments with several amino acids at 25-fold higher levels than spermidine, only methionine, asparagine, and glutamine were effective in reducing uptake of spermidine to 60% of control rates. Based on those observations, this rice gene was named polyamine uptake transporter 1 (OsPUT1). Tissue-specific expression of OsPUT1 by semiquantitative RT-PCR showed that the gene was expressed in all tissues except seeds and roots. Transient expression assays in onion epidermal cells and rice protoplasts failed to localize to a cellular compartment. The characterization of the first plant polyamine transporter sets the stage for a systems approach that can be used to build a model to fully define how the biosynthesis, degradation, and transport of polyamines in plants mediate developmental and biotic responses.

A Screening Method to Identify Essential Yeast Genes for Responses Towards Spermine.

We exploited the yeast DAmP mutant collection to identify essential genes that play a role in polyamine resistance. Herein, we described in details the methodology to obtain these genes. This approach is applicable for screening many nontoxic and toxic drugs.

A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system.

Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verification to determine indels in one or both alleles by Sanger sequencing. This strategy is based on the efficiency of DNA editing, avoids antibiotic selection, and bypasses the need for cell sorting.

The histone H2B Arg95 residue links the pheromone response pathway to rapamycin-induced G1 arrest in yeast.

Rapamycin is an immunosuppressant used for treating many types of diseases such as kidney carcinomas. In yeast, rapamycin inhibits the TORC1 kinase signaling pathway causing rapid alteration in gene expression and ultimately cell cycle arrest in G1 through mechanisms that are not fully understood. Herein, we screened a histone mutant collection and report that one of the mutants, H2B R95A, is strikingly resistant to rapamycin due to a defective cell cycle arrest. We show that the H2B R95A causes defects in the expression of a subset of genes of the pheromone pathway required for α factor-induced G1 arrest. The expression of the STE5 gene and its encoded scaffold protein Ste5, required for the sequential activation of the MAPKs of the pheromone pathway, is greatly reduced in the H2B R95A mutant. Similar to the H2B R95A mutant, cells devoid of Ste5 are also resistant to rapamycin. Rapamycin-induced G1 arrest does not involve detectable phosphorylation of the MAPKs, Kss1, and Fus3, as reported for α factor-induced G1 arrest. However, we observed a sharp induction of the G1 cyclin Cln2 (~ 3- to 4-fold) in the ste5Δ mutant within 30 min of exposure to rapamycin. Our data provide a new insight whereby rapamycin signaling via the Torc1 kinase may exploit the pheromone pathway to arrest cells in the G1 phase.

C. elegans ribosomal protein S3 protects against H2O2-induced DNA damage and suppresses spontaneous mutations in yeast.

Carcinogenicity and cytotoxicity are severe consequences of DNA damage. Base Excision Repair (BER) is a conserved DNA repair pathway that replaces many damaged bases caused by oxidation. Aberrations in BER are associated with carcinogenesis, neurodegeneration, and aging. The nematode C. elegans is an attractive model system for studying BER. However, in this organism, the complete pathway is not fully delineated. To further explore the BER process in C. elegans, we used affinity tag chromatography and mass spectrometry to identify the interactome of uracil DNA glycosylase-1 (CeUNG-1), an enzyme that acts during the first step of the BER pathway. Our analysis identified that CeUNG-1 is associated with the 40 S ribosomal protein S3 (CeRPS-3), homologs of which have been shown to process 8-oxoguanine and abasic site lesions in other organisms. We report a strong in silico association between CeUNG-1 and CeRPS-3 and confirmed this interaction using the yeast two-hybrid system. Downregulation of the Cerps-3 gene reduced the viability of wild-type worms upon exposure to the chemical oxidant hydrogen peroxide. Further analysis shows that Cerps-3 knockdown significantly sensitized the AP endonuclease APN-1-deficient strain, apn-1, but to a lesser extent exo-3, to the lethal effects of hydrogen peroxide. A cross-species complementation experiment reveals that the expression of CeRPS-3 rescued the hydrogen peroxide sensitivity, and suppressed the high mutation frequency of the yeast AP endonuclease-deficient strain lacking Apn1 and Apn2. We propose that CeRPS-3 may function as an auxiliary DNA repair enzyme in C. elegans to process oxidative DNA lesions.

Development of non-viral vectors for neuronal-targeted delivery of CRISPR-Cas9 RNA-proteins as a therapeutic strategy for neurological disorders.

The aging population contributes to an increase in the prevalence of neurodegenerative diseases, such as Parkinson's disease (PD). Due to the progressive nature of these diseases and an incomplete understanding of their pathophysiology, current drugs are inefficient, with a limited efficacy and major side effects. In this study, CRISPR-Cas9 RNA-proteins (RNP) composed of a Cas9 nuclease and single-guide RNA were delivered with a non-viral targeted delivery system to rescue the PD-associated phenotype in neuronal cells. Here, we fused the cell-penetrating amphipathic peptide, PepFect14 (PF14), with a short fragment of the rabies virus glycoprotein (C2) previously shown to have an affinity towards nicotinic acetylcholine receptors expressed on neuronal cells and on the blood-brain barrier. The resultant peptide, C2-PF14, was used to complex with and deliver RNPs to neuronal cells. We observed that RNP/C2-PF14 complexes formed nanosized, monodispersed, and nontoxic nanoparticles that led to a specific delivery into neuronal cells. α-Synuclein (α-syn) plays a major role in the pathology of PD and is considered to be a target for therapy. We demonstrated that CRISPR/Cas9 RNP delivered by C2-PF14 achieved α-syn gene (SNCA) editing in neuronal cells as determined by T7EI assay and western blotting. Furthermore, RNP/C2-PF14 relieved PD-associated toxicity in neuronal cells in vitro. This is a proof-of-concept towards simple and safe targeted genome-editing for treating PD and other neurological disorders.

Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System.

The nuclease activity of the CRISPR-Cas9 system relies on the delivery of a CRISPR-associated protein 9 (Cas9) and a single guide RNA (sgRNA) against the target gene. CRISPR components are typically delivered to cells as either a Cas9/sgRNA ribonucleoprotein (RNP) complex or a plasmid encoding a Cas9 protein along with a sequence-specific sgRNA. Multiple transfection reagents are known to deliver CRISPR-Cas9 components, and delivery vectors are being developed for different purposes by several groups. Here, we repurposed a dual-fluorescence (RFP-GFP-GFP) reporter system to quantify the uptake level of the functional CRISPR-Cas9 components into cells and compare the efficiency of CRISPR delivery vectors. Using this system, we developed a novel and rapid cell-based microplate reader assay that makes possible real-time, rapid, and high throughput quantification of CRISPR nuclease activity. Cells stably expressing this dual-fluorescent reporter construct facilitated a direct quantification of the level of the internalized and functional CRISPR-Cas9 molecules into the cells without the need of co-transfecting fluorescently labeled reporter molecules. Additionally, targeting a reporter gene integrated into the genome recapitulates endogenous gene targeting. Thus, this reporter could be used to optimize various transfection conditions of CRISPR components, to evaluate and compare the efficiency of transfection agents, and to enrich cells containing desired CRISPR-induced mutations.

Genetic engineering to enhance microalgal-based produced water treatment with emphasis on CRISPR/Cas9: A review.

In recent years, the increased demand for and regional variability of available water resources, along with sustainable water supply planning, have driven interest in the reuse of produced water. Reusing produced water can provide important economic, social, and environmental benefits, particularly in water-scarce regions. Therefore, efficient wastewater treatment is a crucial step prior to reuse to meet the requirements for use within the oil and gas industry or by external users. Bioremediation using microalgae has received increased interest as a method for produced water treatment for removing not only major contaminants such as nitrogen and phosphorus, but also heavy metals and hydrocarbons. Some research publications reported nearly 100% removal of total hydrocarbons, total nitrogen, ammonium nitrogen, and iron when using microalgae to treat produced water. Enhancing microalgal removal efficiency as well as growth rate, in the presence of such relevant contaminants is of great interest to many industries to further optimize the process. One novel approach to further enhancing algal capabilities and phytoremediation of wastewater is genetic modification. A comprehensive description of using genetically engineered microalgae for wastewater bioremediation is discussed in this review. This article also reviews random and targeted mutations as a method to alter microalgal traits to produce strains capable of tolerating various stressors related to wastewater. Other methods of genetic engineering are discussed, with sympathy for CRISPR/Cas9 technology. This is accompanied by the opportunities, as well as the challenges of using genetically engineered microalgae for this purpose.

A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor.

The main protease, Mpro, is critical for SARS-CoV-2 replication and an appealing target for designing anti-SARS-CoV-2 agents. Therefore, there is a demand for the development of improved sensors to monitor its activity. Here, we report a pair of genetically encoded, bioluminescence resonance energy transfer (BRET)-based sensors for detecting Mpro proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with Mpro in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in Mpro completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensors developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.

Understanding the Mechanism of Dysglycemia in a Fanconi-Bickel Syndrome Patient.

Fanconi-Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized mainly by the accumulation of glycogen in the liver and kidney. It is inherited as an autosomal recessive disorder caused by mutations in the SLC2A2 gene, which encodes for GLUT2. Patients with FBS have dysglycemia but the molecular mechanisms of dysglycemia are still not clearly understood. Therefore, we aimed to understand the underlying molecular mechanisms of dysglycemia in a patient with FBS. Genomic DNA was isolated from a peripheral blood sample and analyzed by whole genome and Sanger sequencing. CRISPR-Cas9 was used to introduce a mutation that mimics the patient's mutation in a human kidney cell line expressing GLUT2 (HEK293T). Mutant cells were used for molecular analysis to investigate the effects of the mutation on the expression and function of GLUT2, as well as the expression of other genes implicated in dysglycemia. The patient was found to have a homozygous nonsense mutation (c.901C>T, R301X) in the SLC2A2 gene. CRISPR-Cas9 successfully mimicked the patient's mutation in HEK293T cells. The mutant cells showed overexpression of a dysfunctional GLUT2 protein, resulting in reduced glucose release activity and enhanced intracellular glucose accumulation. In addition, other glucose transporters (SGLT1 and SGLT2 in the kidney) were found to be induced in the mutant cells. These findings suggest the last loops (loops 9-12) of GLUT2 are essential for glucose transport activity and indicate that GLUT2 dysfunction is associated with dysglycemia in FBS.