Mechanisms of CRISPR-mediated immunity and applications beyond editing

Mammals, bacteria, and archaea have domesticated transposases (e.g., RAG1 and Cas1) to form adaptive immune systems. Bacteria and archaea acquire resistance to viruses and plasmids by preferentially integrating fragments of foreign DNA at one end of a CRISPR locus. DNA motifs upstream of the CRISPR (i.e., leader) facilitate integration at the first CRISPR repeat. But how do these upstream DNA motifs act over large distances of 130 bp, or roughly 440 A, to regulate integration allosterically? Here, we determine the structure of a 560 KDa integration complex that explains how the CRISPR leader DNA recruits Cas (i.e., Cas1-2/3) and non-Cas proteins (i.e., IHF). Cas1-2/3 and IHF cooperate to fold the genome into a successive U-shaped bend and a loop. The genomic U-bend traps foreign DNA against the integrase, whereas the genomic loop positions the leader-repeat junction at the Cas1 active site. The foreign DNA and the CRISPR repeat wrap around opposite faces of Cas2, poised for a Cas1-catalyzed strand-transfer reaction. The post-integration structure suggests that strand-transfer releases tension in the DNA loop. Therefore Cas1-2/3 may harness protein-induced DNA tension to favor the completion of the isoenergetic integration reaction. Cas1-2/3 interacts extensively with the leader and repeat without making sequence-specific contacts, and we demonstrate that protein-mediated folding of DNA drives integration into diverse sequences. These results reveal Cas1-2/3 and IHF strain DNA to enhance integration allosterically and suggest a mechanism for the de novo generation of new CRISPRs. Further, to address an urgent need for inexpensive and rapid detection of viruses, we recently repurposed a CRISPR immune signaling pathway to detect SARS-CoV-2 in patient samples. A.S-F. is a postdoctoral fellow of the Life Science Research Foundation, supported by the Simons Foundation. A.S-F. is supported by the PDEP award from the Burroughs Wellcome Fund, and by the National Institutes of Health, United States grant 1K99GM147842. This work was also supported by NSF (1828765), NIH (U24 GM129539, R35GM134867).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

cGAS-STING PATHWAY LIMITS SARS-CoV-2 REPLICATION IN ACE2+ AIRWAY CELL LINES

Background: We previously screened 10 human lung and upper airway cell lines expressing variable levels of endogenous ACE2/TMPRSS2. We found that H522 human lung adenocarcinoma cells supported SARS-CoV-2 replication independent of ACE2, whereas the ACE2 positive cell lines were not permissive to infection. Type I/III interferons (IFNs) potently restrict SARS-CoV-2 replication through the actions of hundreds of interferon-stimulated genes (ISGs) that are upregulated upon IFN signaling. Here we report that a number of ACE2 positive airway cell lines are unable to support SARS-CoV-2 replication due to basal activation of the cGAS-STING DNA sensing pathway and subsequent upregulation of IFNs and ISGs which restrict SARS-CoV-2 replication. Method(s): SARS-CoV-2 WT strain 2019-nCoV/USA-WA1/2020 viral replication was detected through analysis of cell associated RNA. RNA sequencing was used to study the basal level of genes in the type-I IFN pathway in the 10 cell lines, which was further validated by western blotting and qRT-PCR. A panel of 5 cell lines, with varying expression levels of ACE2 and TMPRSS2, were pre-treated with Ruxolitinib, a JAK1/2 inhibitor. A siRNA-mediated screen was used to determine the molecular basis of basally high expression of ISGs in cell lines. CRISPR knockout of IFN-alpha receptor and cGAS-STING pathway components was conducted in parallel Results: Here we show that higher basal levels of IFN pathway activity underlie the inability of ACE2+ cell lines to support virus replication. Importantly, this IFN-induced block can be overcome by chemical inhibition and genetic disruption of the IFN signaling pathway or by ACE2 overexpression, suggesting that one or more saturable ISGs underlie the lack of permissivity of these cells. Ruxolitinib treatment increased SARS-CoV-2 RNA levels by nearly 3 logs in OE21 and SCC25. Furthermore, the baseline activation of the STING-cGAS pathway accounts for the high ISG levels and genetic disruption of the cGAS-STING pathway enhances levels by nearly 2 and 3 logs of virus replication in the two separate ACE2+ cell line models respectively. Conclusion(s): Our findings demonstrate that cGAS-STING-dependent activation of IFN-mediated innate immunity underlies the inability of ACE2+ airway cell lines to support SARS-CoV-2 replication. Our study highlights that in addition to ACE2, basal activation of cGAS-STING pathway, IFNs and ISGs may play a key role in defining SARS-CoV-2 cellular tropism and may explain the complex SARS-CoV- 2 pathogenesis in vivo.

Targeting virus-induced vulnerabilities using synthetic lethality as a new class of host-based antivirals

RNA viruses are the major class of human pathogens responsible for many global health crises, including the COVID-19 pandemic. However, the current repertoire of U.S. Food and Drug Administration (FDA)-approved antivirals is limited to only nine out of the known 214 human-infecting RNAviruses, and almost all these antivirals target viral proteins. Traditional antiviral development generally proceeds in a virus-centric fashion, and successful therapies tend to be only marginally effective as monotherapies, due to dose-limiting toxicity and the rapid emergence of drug resistance. Host-based antivirals have potential to alleviate these shortcomings, but do not typically discriminate between infected and uninfected cells, thus eliciting unintended effects. In infected cells where host proteins are repurposed by a virus, normal host protein functions are compromised;a situation analogous to a loss-of-function mutation, and cells harboring the hypomorph have unique vulnerabilities. As well-established in model systems and in cancer therapeutics, these uniquely vulnerable cells can be selectively killed by a drug that inhibits a functionally redundant protein. This is the foundation of synthetic lethality (SL). To test if viral induced vulnerabilities can be exploited for viral therapeutics, we selectively targeted synthetic lethal partners of GBF1, a Golgi membrane protein and a critical host factor for many RNA viruses including poliovirus, Coxsackievirus, Dengue, Hepatitis C and E virus, and Ebola virus. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. A genome-wide chemogenomic CRISPR screen identified synthetic lethal partners of GBF1 and revealed ARF1 as the top hit. Disruption of ARF1, selectively killed cells that synthesize poliovirus 3A alone or in the context of a poliovirus replicon. Combining 3A expression with sub-lethal amounts of GCA - a specific inhibitor of GBF1 further exacerbated the GBF1-ARF1 SL effect. Together our data demonstrate proof of concept for host-based SL targeting of viral infection. We are currently testing all druggable synthetic lethal partners of GBF1 from our chemogenomic CRISPR-screen, in the context of dengue virus infection for their abilities to selectively kill infected cells and inhibit viral replication and infection. Importantly, these SL gene partners of viral-induced hypomorphs only become essential in infected cells and in principle, targeting them will have minimal effects on uninfected cells. Our strategy to target SL interactions of the viral-induced hypomorph has the potential to change the current paradigm for host-based therapeutics that can lead to broad-spectrum antivirals and can be applied to other intracellular pathogens. This work is supported by National Institutes of Health grants R01 GM112108 and P41 GM109824, R21 AI151344 and foundation grant FDN-167277 from the Canadian Institutes of Health Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Reverse Transcription Recombinase Polymerase Amplification integrated with CRISPR Technology and LFA for point-of-care detection of SARS-CoV-2 virus

The field-deployable point-of-care diagnostic test for rapid detection of SARS-COV-2 is needed for implementation of the control measures. In this direction, recently developed CRISPR technology combined with isothermal recombinase polymerase amplification assay is a versatile highly sensitive detection platform for rapid diagnosis of infectious diseases. Here we report the development of RT-RPA-CRISPR based LFA assay for detection of SARS-CoV-2 targeting conserved RdRp and E genes. Various sets of primers and gRNAs were designed targeting conserved regions of the RdRp and E genes of different lineages of SARS-CoV-2 viruses. The isothermal RT-RPA based amplification reactions were standardized using invitro transcribed RNAs of the target regions. The optimum amplification was observed at 42degreeC for 30 min as confirmed by visualization of the amplicons in agarose gel. Subsequently, CRISPRCAS12 reaction was implemented for specific detection of amplicons. Different sets of gRNAs targeting RdRp and E genes were designed and synthesized by in-vitro transcription. The CRISP/CAS12-gRNA complex and single stranded fluorescence probe were added to the RT-RPA amplicons for cleavage of fluorescence probe in positive reaction. Subsequently, the cleaved probes were detected in precoated LFA strips. Upon probe cleavage reaction, the product was mixed with buffer and loaded into LFA strips. In positive reaction, test line showed strong band in test line and light band in control line. The standardized RT-RPA-CRISPR-LFA assay was tested for detection of SARS-CoV-2 using previously isolated RNAs from clinical cases of human SARS-CoV-2 infections. The developed assay successfully detected the positive cases. In conclusion, the developed assay could serve as versatile POC platform for rapid detection of SARS-CoV-2 nucleic acids in human as well as animals.

CRISPR Technology and Its Importance in SARS-CoV-2 Treatment

Coronavirus disease-2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) predominantly affects the respiratory system. The COVID-19 pandemic has had devastating effects on the health system and the global economy worldwide. To reduce the worsening impact of the pandemic, various treatment options and vaccines have been developed. Despite these efforts the pandemic could not be stopped because of the single-stranded nature of the virus combined with the lack of proof-reading abilities of the RNA-dependent RNA polymerase (RdRp). This results in a high probability of error in the copying process and consequently, mutations occur. The increase in mutations in SARS-CoV-2 reduced the efficacy of antiviral medicines and vaccines. To fight this problem, studies were conducted on the efficacy and safety of using Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) in the diagnosis and treatment of COVID-19. Initially, discovered in archaea, CRISPR is a gene-editing tool that works by altering specific parts of the genome. In this review, we focused on the efficacy and safety of CRISPR technology in the treatment of COVID-19.Copyright © 2023 Bilimsel Tip Yayinevi. All rights reserved.

History, Diagnosis, and Treatment of Coronavirus Disease 2019 (COVID-19)

The Coronavirus Disease 2019 (COVID-19), also known as a novel coronavirus (2019-n-CoV), reportedly originated from Wuhan City, Hubei Province, China. Coronavirus Disease 2019 rapidly spread all over the world within a short period. On January 30, 2020, the World Health Organization (WHO) declared it a global epidemic. COVID-19 is a Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) evolves to respiratory, hepatic, gastrointestinal, and neurological complications, and eventually death. SARS-CoV and the Middle East Respiratory Syndrome coron-avirus (MERS-CoV) genome sequences similar identity with 2019-nCoV or Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). However, few amino acid sequences of 2019-nCoV differ from SARS-CoV and MERS-CoV. COVID-19 shares about 90% amino acid sequence simi-larity with SARS-CoV. Effective prevention methods should be taken in order to control this pandemic situation. To date, there are no effective treatments available to treat COVID-19. This review provides information regarding COVID-19 history, epidemiology, pathogenesis and molecular diagnosis. Also, we focus on the development of vaccines in the management of this COVID-19 pandemic and limiting the spread of the virus.Copyright © 2022 Bentham Science Publishers.

COVID-19: From the Molecular Mechanisms to Treatment

The 2019 novel coronavirus (SARS-CoV-2) causes severe pneumonia called COVID-19 and leads to severe acute respiratory syndrome with a high mortality rate. The SARS-CoV-2 virus in the human body leads to jumpstarting immune reactions and multi-organ inflammation, which has poorer outcomes in the presence of predisposing conditions, including hypertension, dyslipidemia, dysglycemia, abnormal adiposity, and even endothelial dysfunction via biomolecular mechanisms. In addition, leucopenia, hypoxemia, and high levels of both cytokines and chemokines in the acute phase of this disease, as well as some abnormalities in chest CT images, were reported in most patients. The spike protein in SARS-CoV-2, the primary cell surface protein, helps the virus anchor and enter the human host cells. Additionally, new mutations have mainly happened for spike protein, which has promoted the infection's transmissibility and severity, which may influence manufactured vaccines' efficacy. The exact mechanisms of the pathogenesis, besides molecular aspects of COVID-19 related to the disease stages, are not well known. The altered molecular functions in the case of immune responses, including T CD4+, CD8+, and NK cells, besides the overactivity in other components and outstanding factors in cytokines like interleukin-2, were involved in severe cases of SARS-CoV-2. Accordingly, it is highly needed to identify the SARS-CoV-2 bio-molecular characteristics to help identify the pathogenesis of COVID-19. This study aimed to investigate the bio-molecular aspects of SARS-CoV-2 infection, focusing on novel SARS-CoV-2 variants and their effects on vaccine efficacy.Copyright © 2022 NRITLD, National Research Institute of Tuberculosis and Lung Disease, Iran.

Assessment of different laboratory tests for the diagnosis of novel coronavirus infections

Rapid diagnosis of coronavirus disease 2019 (COVID-19)-infected patients is urgent in making decisions on public health measures. There are different types of diagnostic tests, such as quantitative PCR assay, antibody, and antigen-based and CRISPR-based tests, which detect genetic materials, viral proteins, or human antibodies in clinical samples. However, the proper test should be highly sensitive, quick, and affordable to address this life-threatening situation. This review article highlights the advantages and disadvantages of each test and compares its different features, such as sensitivity, specificity, and limit of detection to reach a reliable conclusion. Moreover, the FDA- authorized kits and studies' approaches toward these have been compared to provide a better perspective to the researchers.Copyright © 2022 Lippincott Williams and Wilkins. All rights reserved.

5th ECOP, European Conference of Oncology Pharmacy

The proceedings contain 130 papers. The topics discussed include: frequency of measuring body weight in (sub)populations of patients with cancer treated with chemotherapy;simple approach to enhance green tea epigallocatechin gallate stability in aqueous solutions and it bioavailability: experimental and theoretical approaches;incidence of cisplatin-induced nephrotoxicity and associated risk factors: single-center experience;impact of the 2019 coronavirus pandemic on cancer treatment in the Calabria Region, Italy;Palbociclib associated neutropenia in clinical practice;successful introduction of a point mutation into the genome of a primary colon cancer cell line using CRISPR base editing technology;incidence of cisplatin-induced nephrotoxicity and associated risk factors: single-center experience;real world data of alk-inhibitors in patients with advanced or metastic non-small cell lung cancer;pembrolizumab in non-small-cell lung cancer: a systematic review of real life data in Spain;gynecomastia in a male after imatinib treatment for chronic myeloid leukemia;and results after discontinuation of pembrolizumab in metastatic melanoma or lung cancer patients: real-word experience.

Genome editing technology and applications with the type I CRISPR system

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which are representative genome editing technologies, are classified into class 1 and class 2 in terms of evolutionary biology and are further classified into several subtypes. Class 2 CRISPR systems, including type II Cas9 and type V Cas12a, are the most commonly used for genome editing in eukaryotic cells, while type I CRISPR systems within Class 1 are also becoming available. Type I CRISPR recognizes longer target sequences than CRISPR-Cas9 and can induce large deletion mutations of several kilobases. These features demonstrate its potential as a novel and unique genome editing tool that can induce genetic disruption safely and reliably. Thus, it is expected to be utilized for gene therapy and industrial applications. Recently, the DNA cleavage mechanism of type I CRISPR has also revealed details from protein-complex analyses with X-ray crystallography, cryo-electron microscopy, and high-speed atomic force microscopy. The single-strand DNA trans-cleavage activity of type I CRISPR, called collateral activity, has broadened the potential application for CRISPR diagnostics, especially in the development of point-of-care testing methods for COVID-19. In this review, we present an overview of the type I CRISPR system, its application to genome editing, and genetic diagnosis using CRISPR-Cas3.Copyright © 2022

THE UNEXPLORED POSSIBILITIES, PERCEPTIONS AND ETHICAL IMPLICATIONS OF GENE-EDITING ALLERGIC DISEASE: ENGAGING STAKEHOLDERS IN SOUTH AFRICA

Clinically approved cell and gene therapies are opening up future possibilities to treat and prevent myriad diseases, which may include allergic diseases. In South Africa, this could help alleviate the high disease burden and economic cost of treating such diseases. However, even if viable gene-editing options to treat, cure and prevent allergic diseases become safe, effective and affordable for the South African market within the next few decades, the ethical implications and challenges of perceptions, regulation and oversight to ensure safety and equitable access remain. It would be important for all stakeholders involved, including the public and physicians, clinicians and ethicists on clinical and research ethics committees, to be informed about the possibilities, to engage in discussions with one another and to redress any gaps in knowledge. It would be especially important to determine whether cases for gene-editing aimed at allergy would be applied for therapeutic purposes or for enhancement. Much research and discussion remain to be embarked upon;however, it is imperative that research and engagement are expanded and prioritised.

Halting the COVID-19 Widespread: A Audit on the Progresses of Determination, Treatment, and Control Measures

Efforts to prevent the transmission of the SARS-CoV-2 infection are critical in light of the ongoing worldwide spread of COVID-19. Recently developed diagnostic tools include CRISPR, IgG tests, spike protein detection, and artificial intelligence. RT-PCR has been replaced with point-of-care assays, which may be performed at the patient's bedside (RT-PCR). All of these options are available to treat the disease: antivirals and other antiparasitic agents, anti-inflammatory medications like interferon or convalescent plasma, monoclonal antibodies like gamma-globulin, and RNAi treatments like mesenchymal stem cell therapy are among the options (ECMO). More than a dozen different types of vaccines are now being tested in clinical studies. Furthermore, breakthrough technologies that are easily deployable and transportable.In addition, vaccination delivery technologies are being developed. The threat of a second wave of infection needs strict and reasonable control mechanisms to keep mortality to a minimal when governments begin to loosen their lockdown tactics. Research into COVID-19's advances in diagnostics and treatment may serve as a platform for future research that can lead to improved containment strategies. Copyright © 2023 Ubiquity Press. All rights reserved.

A Universal 'Day Zero' Infectious Disease Testing Strategy Exploiting Crispr-Based Sample Depletion And Metagenomic Sequencing

Background. The lack of preparedness for detecting the highly infectious SARS-CoV-2 pathogen - the pathogen responsible for the COVID-19 disease - caused enormous harm to the public health, the economy and society as a whole. It took ~60 days for the first RT-PCR tests for SARS-CoV-2 infection developed by the United States Centers for Disease Control (CDC) to be made available. It then took >270 days to deploy 800,000 of these tests at a time when the estimated actual testing needs required over 6 million tests per day. Testing was therefore limited to only individuals with symptoms or individuals in close contact with confirmed positive cases. Testing strategies that can be deployed on a population scale at 'day zero' (i.e., at the time of the first reported case) are needed. Next Generation Sequencing (NGS) has such day zero capabilities with the potential to enable feasible and broad large-scale testing strategies, however it has limited detection sensitivity for low copy numbers of pathogens which may be present. Here we demonstrate that using CRISPR-Cas9 to remove abundant sequences that do not contribute to pathogen detection, NGS detection sensitivity is equivalent to RT-PCR. In addition, we show that this assay can be used for variant strain typing, co-infection detection, and individual human host response assessment - all in a single workflow using existing open-source analysis pipelines. This NGS workflow is pathogen agnostic, and therefore has the potential to radically transform how both very large-scale pandemic response and focused clinical infectious disease testing are pursued in the future. Methods. Covid positive samples with RT-PCR Ct values from 16-39 were processed through the CRISRP enhanced mNGS pipeline. Results. Sn/Sp compared to RT-PCR was 97%/100%. Strain calling concordance compared to amplicon sequencing was 100%. Co-infections from Covid positive samples were identified with high confidence. Host response signatures match the published literature. Conclusion. Applying CRISPR enhanced metagenomic NGS at Day Zero of the next pandemic can mitigate the time gap in developing approved diagnostics at population scale and potentially save lives.

Aberrant DNA 5mC and 6mA methylations increase ACE2 expression in intestinal cancer cells susceptible to SARS-CoV-2 infection

Angiotensin converting enzyme II (ACE2) is the cellular receptor of SARS-CoV-2. At present, ACE2 receptor is considered to be the key component in the SARS-CoV-2 infection and transmitting in the host. Among the cancer patients with COVID-19, the gastrointestinal cancer is the second most prevalent. The MethyLight and QASM assays were used to evaluated the genomic DNA 5mC methylation, while the CviAII enzyme-based 6mA-RE-qPCR was applied to determine motif-specific DNA 6mA methylation. The 6mA and 5mC methylation analyses of the long interspersed nuclear elements 1 (LINE1) were used to evaluate the global level of genomic 6mA and 5mC methylations, respectively. To investigate the role of ACE2 DNA methylation in regulating ACE2 expression, we performed a genome-wide methylation analysis in colorectal cancer samples collected at the Sixth Affiliated Hospital of Sun Yat-sen University. The DNA 5mC methylation of ACE2 promoter in tumor tissues were significantly lower than that in normal tissues, while the DNA 6mA methylation of ACE2 promoter in tumor tissues was significantly higher than that in normal tissues. In addition, the mRNA and protein expression of ACE2 in tumor tissues were lower than that in normal tissues. To explore the epigenetic regulation on ACE2 expression, we treated colon cancer cell lines with 5-Azacytidine and found ACE2 expression was upregulated after lowering the DNA 5mC methylation. The correlation analysis in patient cohort samples showed that ACE2 mRNA expression was positively correlated with DNA 5mC and negatively associated with DNA 6mA methylation. Next, a novel CRISPR-based tool was developed for sequence-specific 6mA editing on ACE2 promoter region, and it was applied in HCT116 cell to further confirm the regulatory role of DNA 6mA methylation in ACE2 mRNA expression. This tool was proved to be reliable with our findings that the CRISPR/dCas9-METTL3 tool could dramatically upregulate DNA 6mA methylation in ACE2 promoter, while the global level of genomic 6mA methylation remained unchanged. Both the mRNA and protein expression of ACE2 were significantly increased following a sequence-specific DNA 6mA editing in ACE2 promoter. In conclusion, we revealed the aberrant DNA 5mC and 6mA methylations in colorectal cancer, which upregulate ACE2 expression in colorectal cancer cells that may confer the susceptibility to SARS-CoV-2 infection. We developed a novel CRISPR-based tool that could realize site-directed 6mA methylation editing. Notably, the epigenetic regulation of DNA 6mA methylation on ACE2 expression provides an insight into the intersection of the biology of cancer, SARS-CoV-2 infection and organ-specific complication in COVID-19. Aberrant ACE2 methylation may serve as a biomarker and treatment target in these patients.

CRISPR gene editing: a revolution in progress

CRISPR is a gene editing technique that has revolutionised research and has the potential to transform the treatment of many diseases. This article discusses the principles of the technique, its therapeutic applications and potential safety issues.

MOLECULAR DETERMINANTS of SARS-CoV-2 CELLULAR TROPISM in VITRO

Background: Lung cell lines to model SARS-CoV-2 replication in vitro are greatly limited hampering the rigorous study of SARS-CoV-2-host interactions. We analyzed a panel of 10 airway cell lines with various levels of ACE2 expression to identify models of SARS-CoV-2 infection. We found that none of the ACE2 expressing cell lines supported replication, whereas the H522 human lung adenocarcinoma cells were naturally permissive to SARS-CoV-2 infection despite detectable expression of ACE2. We confirmed that SARS-CoV-2 replication is indeed completely independent of ACE2 in H522s but dependent on heparan sulfates and the E484D substitution within the Spike. Further, we show that many of the ACE2 positive non-permissive cell lines express high basal levels of interferon-stimulated genes, which can be overcome by inhibition of the JAK/STAT pathway or by ACE2 overexpression. Together, our findings highlight ACE2-independent pathways can control the cellular tropism of SARS-CoV-2. Methods: Conventional molecular virology assays have been conducted to study the permissiveness of a panel of 10 cell lines expressing various levels of ACE2. ACE2 independence of SARS-CoV-2 replication was validated by antibody blocking, Fc-ACE2 decoy peptide and CRISPR-based approaches in H522 cells. RNA sequencing was used to study the basal level of genes in the type-I IFN pathway in the panel of 10 cell lines, which was further validated by western blotting and qRT-PCR. A panel of 5 cell lines, with varying expression levels of ACE2 and TMPRSS2, were pre-treated with Ruxolitinib, a JAK inhibitor, and infected with SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 and spike variants. Viral replication was detected through analysis of cell associated RNA Results: H522 human lung adenocarcinoma supports SARS-CoV-2 replication in a completely ACE2-independent manner. Transcriptomic analysis revealed basal high level of expression of interferon response pathway genes in some ACE2-positive cells recalcitrant to SARS-CoV-2 infection. Infection of OE21 and SCC25 cells required blocking of the IFN response pathway or ACE2 overexpression to allow SARS-CoV-2 infection. Conclusion: These findings suggest that SARS-CoV-2 replication can proceed in complete absence of ACE2 and that the innate immunity is a key determinant of SARS-CoV-2 cellular tropism. These findings may explain the complex SARS-CoV-2 pathogenesis in vivo as it shows that factors independent of ACE2 can define cellular tropism.

SARS-CoV-2 origin, myths and diagnostic technology developments

Background: After the first case of COVID-19 being announced in China in December 2019, various diagnostic technologies have been developed at unprecedented pace with the aim of providing a basis for accurate clinical intervention. However, some assays including CRISPR-based diagnostics and loop-mediated isothermal amplification (LAMP) have been less explored. As new COVID-19 technologies emerge, there is need for them to be assessed, validated and improved upon. Moreover, there is paucity of data on the essential factors governing the selection of an appropriate diagnostic approach within the correct timeframe. Myths and origin of SARS-CoV-2 remain to be controversial. Consequently, this review aims at exploring the current COVID-19 diagnostic technologies, performance evaluation, principles, suitability, specificity, sensitivity, successes and challenges of the technologies for laboratory and bedside testing. Main Body: To date, there exist more publications on COVID-19 diagnostics as compared to the Zika virus. The SARS-CoV-2 virus genome profiles were readily available by 31st of December 2019. This was attributed to the fast-paced sharing of the epidemiological and diagnostics data of COVID-19. Timely profiling of the virus genome accelerated the development of diagnostic technologies. Furthermore, the rapid publication of studies that evaluated several diagnostic methods available provided baseline information on how the various technologies work and paved way for development of novel technologies. Conclusion: Up to date, RT-PCR is the most preferred as compared to the other assays. This is despite the repeated false negatives reported in many of the study findings. Considering that COVID-19 has caused devastating effects on the economy, healthcare systems, agriculture and culture, timely and accurate detection of the virus is paramount in the provision of targeted therapy hence reducing chances of drug resistance, increased treatment costs and morbidity. However, information on the origin of SARS-CoV-2 still remains elusive. Furthermore, knowledge and perception of the patients toward management of SARS-CoV-2 are also paramount to proper diagnosis and management of the pandemic. Future implications of the misperceptions are that they may lead to increased non-compliance to SARS-CoV-2-related World Health Organization (WHO) policies and guidelines.