Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection

Background The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics. Methods We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system. We validated both ENHANCE and ENHANCEv2 using 62 nasopharyngeal swabs and compared the results to RT-qPCR. We created a lyophilized version of ENHANCEv2 and characterized its detection capability and stability. Results Here we demonstrate that when coupled with an RT-LAMP step, ENHANCE detects COVID-19 samples down to a few copies with 95% accuracy while maintaining a high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. ENHANCE works robustly in a wide range of magnesium concentrations (3 mM-13 mM), allowing for further assay optimization. Our clinical validation results for both ENHANCE and ENHANCEv2 show 60/62 (96.7%) sample agreement with RT-qPCR results while only using 5 µL of sample and 20 minutes of CRISPR reaction. We show that the lateral flow assay using paper-based strips displays 100% agreement with the fluorescence-based reporter assay during clinical validation. Finally, we demonstrate that a lyophilized version of ENHANCEv2 shows high sensitivity and specificity for SARS-CoV-2 detection while reducing the CRISPR reaction time to as low as 3 minutes while maintaining its detection capability for several weeks upon storage at room temperature. Conclusions CRISPR-based diagnostic platforms offer many advantages as compared to conventional qPCR-based detection methods. Our work here provides clinical validation of ENHANCE and its improved form ENHANCEv2 for the detection of COVID-19.

Validation of Specific and Reliable Genetic Tools to Identify, Label, and Target Cardiac Pericytes in Mice

Background In the myocardium, pericytes are often confused with other interstitial cell types, such as fibroblasts. The lack of well‐characterized and specific tools for identification, lineage tracing, and conditional targeting of myocardial pericytes has hampered studies on their role in heart disease. In the current study, we characterize and validate specific and reliable strategies for labeling and targeting of cardiac pericytes. Methods and Results Using the neuron‐glial antigen 2 (NG2) DsRed reporter line, we identified a large population of NG2+ periendothelial cells in mouse atria, ventricles, and valves. To examine possible overlap of NG2+ mural cells with fibroblasts, we generated NG2 DsRed ; platelet‐derived growth factor receptor (PDGFR) α EGFP pericyte/fibroblast dual reporter mice. Myocardial NG2+ pericytes and PDGFRα+ fibroblasts were identified as nonoverlapping cellular populations with distinct transcriptional signatures. PDGFRα+ fibroblasts expressed high levels of fibrillar collagens, matrix metalloproteinases, tissue inhibitor of metalloproteinases, and genes encoding matricellular proteins, whereas NG2+ pericytes expressed high levels of Pdgfrb , Adamts1 , and Vtn . To validate the specificity of pericyte Cre drivers, we crossed these lines with PDGFRα EGFP fibroblast reporter mice. The constitutive NG2 Cre driver did not specifically track mural cells, labeling many cardiomyocytes. However, the inducible NG2 CreER driver specifically traced vascular mural cells in the ventricle and in the aorta, without significant labeling of PDGFRα+ fibroblasts. In contrast, the inducible PDGFRβ CreER line labeled not only mural cells but also the majority of cardiac and aortic fibroblasts. Conclusions Fibroblasts and pericytes are topographically and transcriptomically distinct populations of cardiac interstitial cells. The inducible NG2 CreER driver optimally targets cardiac pericytes; in contrast, the inducible PDGFRβ CreER line lacks specificity.

A dual reporter system identifies an intermediate state and sequential regulators of 2-cell-like-to-pluripotent state transition

Mouse embryonic stem cells (ESCs) cycle in and out of 2-cell-like (2C-like) state in culture. The molecular mechanism governing the exit of 2C-like state remains obscure, partly due to the lack of a reporter system that can genetically mark intermediate states during exiting process. Here, we identify an intermediate state that is marked by the co-expression of MERVL::tdTomato and OCT4-GFP (MERLOT) during 2C-like-to-pluripotent state transition (2CLPT). Transcriptome and epigenome analyses demonstrate that MERLOT cells cluster closely with 8-16 cell stage mouse embryos, suggesting that 2CLPT partly mimics early preimplantation development. Through a CRISPRa screen, we identify an ARRDC3-NEDD4-OCT4 regulatory axis that plays an essential role in controlling 2CLPT. Furthermore, re-evaluating previously reported 2C-like state regulators reveals dual function of Chaf1a in regulating the entry and exit of 2C-like state. Finally, ATAC-Seq footprinting analysis uncovers Klf3 as an essential transcription factor required for efficient 2CLPT. Together, our study identifies a genetically traceable intermediate state during 2CLPT and provides a valuable tool to study molecular mechanisms regulating this process.

A Dual-reporter Platform for Screening Tumor-targeted Extracellular Vesicles

Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and may improve targeted drug delivery. In the past decade, various approaches to EV surface modification for targeting specific cells or tissues have been proposed, including genetic engineering of parental cells or postproduction EV engineering. However, due to technical limitations, targeting moieties of engineered EVs have not been thoroughly characterized. Here, we report the bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL-based dual-reporter platform for characterizing the cellular uptake of tumor homing peptide (THP)-engineered EVs, targeting PDL1, uPAR, or EGFR proteins expressed in MDA-MB-231 breast cancer cells, simultaneously by bioluminescence measurement and fluorescence microscopy. Bioluminescence analysis of cellular EV uptake revealed the highest binding efficiency of uPAR-targeted EVs, whereas PDL1-targeted EVs showed slower cellular uptake. EVs engineered with two known EGFR-binding peptides via lipid nanoprobes did not increase cellular uptake, indicating that designs of EGFR-binding peptide conjugation to the EV surface are critical for functional EV engineering. Fluorescence analysis of cellular EV uptake allowed us to track individual PalmReNL-EVs bearing THPs in recipient cells. These results demonstrate that the PalmReNL-based EV assay platform can be a foundation for high-throughput screening of tumor-targeted EVs.

Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e. tachyzoites) and for establishing chronic infection (i.e. bradyzoites). Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca2+ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of Ca2+-ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca2+ rapidly restored their intracellular Ca2+ and ATP stores leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.

Development of a Single-cycle Infectious SARS-CoV-2 Virus Replicon Particle System for use in BSL2 Laboratories

Research activities with infectious severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) are currently permitted only under biosafety level 3 (BSL3) containment. Here, we report the development of a single-cycle infectious SARS-CoV-2 virus replicon particle (VRP) system with a luciferase and green fluorescent protein (GFP) dual reporter that can be safely handled in BSL2 laboratories to study SARS-CoV-2 biology. The Spike (S) gene of SARS-CoV-2 encodes for the envelope glycoprotein, which is essential for mediating infection of new host cells. Through deletion and replacement of this essential S gene with a luciferase and GFP dual reporter, we have generated a conditional SARS-CoV-2 mutant (ΔS-VRP) that produces infectious particles only in cells expressing a viral envelope glycoprotein of choice. Interestingly, we observed more efficient production of infectious particles in cells expressing vesicular stomatitis virus (VSV) glycoprotein G (ΔS-VRP(G)) as compared to cells expressing other viral glycoproteins including S. We confirmed that infection from ΔS-VRP(G) is limited to a single round and can be neutralized by anti-VSV serum. In our studies with ΔS-VRP(G), we observed robust expression of both luciferase and GFP reporters in various human and murine cell types, demonstrating that a broad variety of cells can support intracellular replication of SARS-CoV-2. In addition, treatment of ΔS-VRP(G) infected cells with anti-CoV drugs remdesivir (nucleoside analog) or GC376 (CoV 3CL protease inhibitor) resulted in a robust decrease in both luciferase and GFP expression in a drug-dose and cell-type dependent manner. Taken together, we have developed a single-cycle infectious SARS-CoV-2 VRP system that serves as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high throughput screening of antiviral drugs under BSL2 containment. Importance Due to the highly contagious nature of SARS-CoV-2 and the lack of immunity in the human population, research on SARS-CoV-2 has been restricted to biosafety level 3 laboratories. This has greatly limited participation of the broader scientific community in SARS-CoV-2 research and thus has hindered the development of vaccines and antiviral drugs. By deleting the essential Spike gene in the viral genome, we have developed a conditional mutant of SARS-CoV-2 with luciferase and fluorescent reporters, which can be safely used under biosafety level 2 conditions. Our single-cycle infectious SARS-CoV-2 virus replicon system can serve as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high throughput screening of antiviral drugs under BSL2 containment.

Quantitative spatial and temporal assessment of regulatory element activity in zebrafish

Mutations or genetic variation in noncoding regions of the genome harbouring cis-regulatory elements (CREs), or enhancers, have been widely implicated in human disease and disease risk. However, our ability to assay the impact of these DNA sequence changes on enhancer activity is currently very limited because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type and disease-associated mutant human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. The activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing simultaneous visualisation of where and when in development the wild-type allele is active and how this activity is altered by mutation.

CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids

Human pluripotent stem cells (PSCs) represent a powerful tool to investigate human eye development and disease. When grown in 3D, they can self-assemble into laminar organized retinas; however, variation in the size, shape and composition of individual organoids exists. Neither the microenvironment nor the timing of critical growth factors driving retinogenesis are fully understood. To explore early retinal development, we developed a SIX6-GFP reporter that enabled the systematic optimization of conditions that promote optic vesicle formation. We demonstrated that early hypoxic growth conditions enhanced SIX6 expression and promoted eye formation. SIX6 expression was further enhanced by sequential inhibition of Wnt and activation of sonic hedgehog signaling. SIX6 + optic vesicles showed RNA expression profiles that were consistent with a retinal identity; however, ventral diencephalic markers were also present. To demonstrate that optic vesicles lead to bona fide “retina-like” structures we generated a SIX6-GFP/POU4F2-tdTomato dual reporter line that labeled the entire developing retina and retinal ganglion cells, respectively. Additional brain regions, including the hypothalamus and midbrain-hindbrain (MBHB) territories were identified by harvesting SIX6 + /POU4F2- and SIX6- organoids, respectively. Using RNAseq to study transcriptional profiles we demonstrated that SIX6-GFP and POU4F2-tdTomato reporters provided a reliable readout for developing human retina, hypothalamus, and midbrain/hindbrain organoids.

764 Characterization of RVU-27065 a novel small-molecule STING agonist suitable for systemic administration

BackgroundSTimulator of INterferon Genes (STING) is a key signaling protein involved in activation of the immune system in response to self-DNA. In recent years, STING signaling has been demonstrated to play a major role in activating the antitumor immune response and therefore is considered an attractive drug target in immuno-oncology. The first wave of STING agonists, cyclic-dinucleotide analogues of the internal ligand cGAMP, were developed for local, intratumoral administration. Herein we present the most recent profiling results of our frontrunner molecule RVU-27065, a potent and selective systemic STING agonist with a favorable drug profile.MethodsBinding to recombinant STING protein was examined using Fluorescence Thermal Shift and Fluorescence Polarisation. Primary activity screen was performed in THP-1 Dual reporter cells. Selectivity was confirmed in THP-1 reporter cells with knocked out STING or expressing STING variants. T cell viability and proliferation was assessed by flow cytometry using activated human T cells. PBMCs were isolated by density gradient from whole blood of healthy donors. Downstream STING pathway activation in cells treated with RVU-27065 was confirmed using Western blot analysis. BALB/c mice were inoculated with EMT6 tumor cells and the compound was administered intravenously followed by regular monitoring of tumor growth. Cured animals were rechallenged by repeated inoculation of EMT6 cells.ResultsRVU-27065 binds and strongly thermostabilizes recombinant STING proteins of all tested species. Binding to the protein results in activation of downstream signalling pathway, confirmed by western blot analysis. The agonist is characterized by selectivity and excellent potency in THP-1 dual reporter cells as well as in human PBMCs and dendritic cells. Short term incubation of RVU-27065 has no impact on T cell viability, activation or proliferation. Furthermore, STING activation with RVU-27065 leads to repolarization of immunosuppressive M2 macrophages into pro-inflammatory M1-like phenotype. In vivo efficacy of RVU-27065 was confirmed, leading to significant tumor growth inhibition and complete tumor regressions in an EMT6 mouse breast cancer syngeneic tumor model.ConclusionsRVU-27065 is a novel representative of a 3rd generation of Ryvu STING agonists – small-molecule, non-macrocyclic molecules built around a unique chemotype. The compound is characterized by high in vitro potency which translates to efficacy in vivo in preclinical animal models. Drug-like properties, excellent selectivity and a good safety profile make RVU-27065 an attractive candidate for further development for standalone as well as targeted delivery, which holds high potential for improved immunotherapy in cancer patients.