Enabling mRNA medicine for brain tumors

mRNA is a new class of drugs that has the potential to revolutionize the treatment of brain tumors. Thanks to the COVID-19 mRNA vaccines and numerous therapy-based clinical trials, it is now clear that lipid nanoparticles (LNPs) are a clinically viable means to deliver RNA therapeutics. However, LNP-mediated mRNA delivery to brain tumors remains elusive. Over the past decade, numerous studies have shown that tumor cells communicate with each other via small extracellular vesicles, which are around 100 nm in diameter and consist of lipid bilayer membrane similar to synthetic lipidbased nanocarriers. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain tumors without undue toxicity. We synthesized LNPs using four components similar to the formulation used in the mRNA COVID19 vaccines (Moderna and Pfizer): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. For the in vitro screen, we tested ten classes of helper lipids based on their abundance in extracellular vesicle membranes, commercial availability, and large-scale production feasibility while keeping rest of the LNP components unchanged. The transfection kinetics of GFP mRNA encapsulated in LNPs and doped with 16 mol% of helper lipids was tested using GL261, U87 and SIM-A9 cell lines. Several LNP formations resulted in stable transfection (upto 5 days) of GFP mRNA in all the cell lines tested in vitro. The successful LNP candidates (enabling >80% transfection efficacy) were then tested in vivo to deliver luciferase mRNA to brain tumors via intrathecal administration in a syngeneic glioblastoma (GBM) mouse model, which confirmed luciferase expression in brain tumors in the cortex. LNPs were then tested to deliver Cre recombinase mRNA in syngeneic GBM mouse model genetically modified to express tdTomato under LoxP marker cassette that enabled identification of LNP targeted cells. mRNA was successfully delivered to tumor cells (70-80% transfected) and a range of different cells in the tumor microenvironment, including tumor-associated macrophages (80-90% transfected), neurons (31- 40% transfected), neural stem cells (39-62% transfected), oligodendrocytes (70-80% transfected) and astrocytes (44-76% transfected). Then, LNP formulations were assessed for delivering Cas9 mRNA and CD81 sgRNA (model protein) in murine syngeneic GBM model to enable gene editing in brain tumor cells. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~94% of brain tumor cells in vivo. In conclusion, we have developed a library of safe LNPs that can transfect GBM cells in vivo with high efficacy. This technology can potentially be used to develop novel mRNA therapies for GBM by delivering single or multiple mRNAs and holds great potential as a tool to study brain tumor biology.

Mesenchymal Stem/Stromal Cells: DEVELOPMENT OF A GMP BANKING PROCESS FOR UMBILICAL CORD MESENCHYMAL STROMAL CELLS (UC-MSCS) AND USE OF UC-MSC IN CHARACTERISATION ASSAYS, FUNCTIONAL ASSAYS AND GENE EDITING STUDIES

Background & Aim: The pro-angiogenic, immunoregulatory and anti- inflammatory properties of MSCs are being exploited for the development of cellular therapies, including the treatment of graft versus host disease (GvHD), inflammatory bowel disease and COVID-19. SNBTS have developed a GMP process to bank umbilical cord MSCs (UC-MSCs) whereby we can reliably bank 100 vials of 10 million P2 UC-MSCs per cord. Each of these vials can be extensively expanded and stored for specific applications. The ultimate aim of the bank is for off-the-shelf clinical use, e.g., in GvHD or as an adjuvant therapy in Islet transplantations. Methods, Results & Conclusion(s): During process development, different basal media and supplements were screened for proliferation and MSC marker expression. Cells grown in promising media combinations were then tested for tri-lineage differentiation (identity), their chemokine/cytokine expression and T-cell inhibition (function) assessed. Medium selected for further GMP development and scale up was ultimately determined by all round performance and regulatory compliance. GMP-like UC-MSCs were shown to have immune-modulatory activity in T-cell proliferation assays at 4:1 or 16:1 ratios. Co-culture of UC-MSCs and freshly isolated leukocytes, +/- the immune activating agent LPS, show a dose dependent survival effect on leukocytes. In particular, neutrophils, which are normally very short lived in vitro demonstrated increased viability when co-cultured with UCMSCs. The survival effect was partially reproduced when UC-MSC were replaced with conditioned medium or cell lysate indicating the involvement of soluble factors. This improved neutrophil survival also correlates with results from leukocyte migration studies that demonstrate neutrophils to be the main cell type attracted to MSCs in in vitro and in vivo. Genetic modification of UC-MSC may improve their therapeutic potential. We have tested gene editing by CRISPR/Cas9 technology in primary UC-MSCS. The CXCL8 gene, highly expressed in UC-MSC, was targeted in isolates from several different donors with editing efficiencies of 78-96% observed. This translated to significant knockdown of CXCL8 protein levels in resting cells, however after stimulation levels of CXCL8 were found to be very similar in edited and non-edited UC-MSCs. This observation requires further study, but overall the results show the potential to generate future banks of primary UC-MSCS with genetically enhanced pro-angiogenic, immunoregulatory and/or anti-inflammatory activities.Copyright © 2023 International Society for Cell & Gene Therapy

A Simple, Pipette-free, and Power-free Device for Virus Nucleic Acid Detection

A portable, inexpensive, and easy-to-manufacture microfluidic device is developed for the detection of SARS-CoV-2 dsDNA fragments. In this device, four reaction chambers separated by carbon fiber rods are pre-loaded with isothermal amplification and CRISPR-Cas12a reagents. The reaction is carried out by simply pulling the rods, without the need for manual pipetting. To facilitate power-free pathogen detection, the entire detection is designed to be heated with a disposable hand warmer. After the CRISPR reaction, the fluorescence signal generated by positive samples is identified by naked eye, using an inexpensive flashlight. This simple and sensitive device will serve as a new model for the next-generation viral diagnostics in either hospital or resource-limited settings. © 2023 SPIE.

Evaluation of the immunogenicity of auxotrophic Lactobacillus with CRISPR-Cas9D10A system-mediated chromosomal editing to express porcine rotavirus capsid protein VP4.

Porcine rotavirus (PoRV) is an important pathogen, leading to the occurrence of viral diarrhoea . As the infection displays obvious enterotropism, intestinal mucosal immunity is the significant line of defence against pathogen invasion. Moreover, as lactic acid bacteria (LAB) show acid resistance, bile salt resistance and immune regulation, it is of great significance to develop an oral vaccine. Most traditional plasmid delivery vectors use antibiotic genes as selective markers, easily leading to antibiotic accumulation. Therefore, to select a food-grade marker in genetically engineering food-grade microorganisms is vital. Based on the CRISPR-Cas9D10A system, we constructed a stable auxotrophic Lactobacillus paracasei HLJ-27 (Lactobacillus △Alr HLJ-27) strain. In addition, as many plasmids replicate in the host bacteria, resulting in internal gene deletions. In this study,we used a temperature-sensitive gene editing plasmidto insert the VP4 gene into the genome, yielding the insertion mutant strains VP4/△Alr HLJ-27, VP4/△Alr W56, and VP4/W56. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses. These oral mucosal vaccines have the potential to act as an alternative to the application of antibiotics in the future and induce efficient immune responses against PEDV infection.

Framework-Hotspot Enhanced Trans Cleavage of CRISPR-Cas12a for Clinical Samples Detection.

The trans-cleavage property of CRISPR-Cas12a system makes it an excellent tool for disease diagnosis. Nevertheless, most methods based on CRISPR-Cas system still require pre-amplification of the target to achieve the desired detection sensitivity. Here we generate Framework-Hotspot reporters (FHRs) with different local densities to investigate their effect on trans-cleavage activity of Cas12a. We find that the cleavage efficiency increases and the cleavage rate accelerates with increasing reporter density. We further construct a modular sensing platform with CRISPR-Cas12a-based target recognition and FHR-based signal transduction. Encouragingly, this modular platform enables sensitive (100 fM) and rapid (<15 min) detection of pathogen nucleic acids without pre-amplification, as well as detection of tumor protein markers in clinical samples. The design provides a facile strategy for enhanced trans cleavage of Cas12a, which accelerates and broadens its applications in biosensing.

Evidence of Copper Nanoparticles and Poly I:C Modulating Cas9 Interaction and Cleavage of COR (Conserved Omicron RNA).

Conserved omicron RNA (COR) is a 40 base long 99.9% conserved sequence in SARS-CoV-2 Omicron variant, predicted to form a stable stem loop, the targeted cleavage of which can be an ideal next step in controlling the spread of variants. The Cas9 enzyme has been traditionally utilized for gene editing and DNA cleavage. Previously Cas9 has been shown to be capable of RNA editing under certain conditions. Here we investigated the ability of Cas9 to bind to single-stranded conserved omicron RNA (COR) and examined the effect of copper nanoparticles (Cu NPs) and/or polyinosinic-polycytidilic acid (poly I:C) on the RNA cleavage ability of Cas9. The interaction of the Cas9 enzyme and COR with Cu NPs was shown by dynamic light scattering (DLS) and zeta potential measurements and was confirmed by two-dimensional fluorescence difference spectroscopy (2-D FDS). The interaction with and enhanced cleavage of COR by Cas9 in the presence of Cu NPs and poly I:C was shown by agarose gel electrophoresis. These data suggest that Cas9-mediated RNA cleavage may be potentiated at the nanoscale level in the presence of nanoparticles and a secondary RNA component. Further explorations in vitro and in vivo may contribute to the development of a better cellular delivery platform for Cas9.

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application.

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.

Multi-faceted CRISPR/Cas technological innovation aspects in the framework of 3P medicine.

Since 2009, the European Association for Predictive, Preventive and Personalised Medicine (EPMA, Brussels) promotes the paradigm change from reactive approach to predictive, preventive, and personalized medicine (PPPM/3PM) to protect individuals in sub-optimal health conditions from the health-to-disease transition, to increase life-quality of the affected patient cohorts improving, therefore, ethical standards and cost-efficacy of healthcare to great benefits of the society at large. The gene-editing technology utilizing CRISPR/Cas gene-editing approach has demonstrated its enormous value as a powerful tool in a broad spectrum of bio/medical research areas. Further, CRISPR/Cas gene-editing system is considered applicable to primary and secondary healthcare, in order to prevent disease spread and to treat clinically manifested disorders, involving diagnostics of SARS-Cov-2 infection and experimental treatment of COVID-19. Although the principle of the proposed gene editing is simple and elegant, there are a lot of technological challenges and ethical considerations to be solved prior to its broadly scaled clinical implementation. This article highlights technological innovation beyond the state of the art, exemplifies current achievements, discusses unsolved technological and ethical problems, and provides clinically relevant outlook in the framework of 3PM.

CRISPR-Cas12a-assisted elimination of the non-specific signal from non-specific amplification in the Exponential Amplification Reaction.

Non-specific amplification is a major problem in nucleic acid amplification resulting in false-positive results, especially for exponential amplification reactions (EXPAR). Although efforts were made to suppress the influence of non-specific amplification, such as chemical blocking of the template's 3'-ends and sequence-independent weakening of template-template interactions, it is still a common problem in many conventional EXPAR reactions. In this study, we propose a novel strategy to eliminate the non-specific signal from non-specific amplification by integrating the CRISPR-Cas12a system into two-templates EXPAR. An EXPAR-Cas12a strategy named EXPCas was developed, where the Cas12a system acted as a filter to filter out non-specific amplificons in EXPAR, suppressing and eliminating the influence of non-specific amplification. As a result, the signal-to-background ratio was improved from 1.3 to 15.4 using this method. With microRNA-21 (miRNA-21) as a target, the detection can be finished in 40 min with a LOD of 103 fM and no non-specific amplification was observed.

CriSPR as a novel technique for COVID-19 diagnosis: a review

To this moment, the human coronavirus disease COVID-19 that occurs as a result of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is still a critical case that provokes concern around the world. In January 2022, there were over 300 million infections and over 5 million fatalities from COVID-19. As a countermeasure against this rapid spread, there is a vital need for effective and low-cost diagnosis methods in order to control the danger of this pandemic. CRISPR technology has proved its ef-ficiency in detecting COVID-19 due to its simplicity, specificity and high sensitivity. This paper reviews the state-of-the-art of developing the CRISPR platforms for the purpose of COVID-19 diagnosis and treatment. Limitations and challenges of CRISPR in terms of nucleic acid analytical methods for viral infection diagnosis are discussed. © 2023, Palladin Institute of Biochemistry of the NASU. All rights reserved.

Optimal Stabilization for Long-Term Storage of Nucleic Acid-Based CRISPR/Cas12a Assay for SARS-CoV-2 Detection

In this long-term storage study, we optimized the lyophilization conditions of each reaction stage of a nucleic acid-based assay for SARS-CoV-2 detection. The stability testing demonstrated that the dried reactions from all 3 steps (cDNA synthesis, isothermal amplification and detection) can be kept at ¡20 °C or 4 °C for up to 6 or 3 months, respectively, whereas, if stored at 25 °C or 37 °C, the reagents only could be stored for a few days without quality loss. This suggests that we can have the dried reactions at ¡20 °C for long-term storage until needed. Moreover, this assay is now simpler to perform as each of the 3 steps now proceeds with pre-mixed regents lyophilized in a single tube for each step. © 2023 University of Kerbala. All rights reserved.

Recent advances on CRISPR/Cas system-enabled portable detection devices for on-site agri-food safety assay

Agri-food safety has been considered as one of the most important public concerns worldwide. From farm to table, food crops and foods are extremely vulnerable to the contamination by a variety of pollutants from their growth and processing. Moreover, the SARS-CoV-2 detected in the food supply chain during COVID-19 pandemic has posed a greater challenge for rapid and on-site detection of agri-food contaminants in complex and volatile environments. Therefore, the development of rapid, accurate, and on-site detection technologies and portable detection devices is of great importance to ensure the agri-food security. This review comprehensively summarized the recent advances on the construction of CRISPR/Cas systems-based biosensing technologies and their portable detection devices, as well as their promising applications in the field of agri-food safety. First of all, the classification and working principles of CRISPR/Cas systems were introduced. Then, the latest advances on the CRISPR/Cas system-based on-site detection technologies and portable detection devices were also systematically summarized. Most importantly, the state-of-the-art applications of CRISPR/Cas systems-based on-site detection technologies and portable detection devices in the fields of agri-food safety were comprehensively summarized. Impressively, the future opportunities and challenges in this emerging and promising field were proposed. Emerging CRISPR/Cas system-based on-site detection technologies have showed a great potential in the detection of agri-food safety. Impressively, the integration of CRISPR/Cas systems-based biosensing technologies with portable detection devices (e.g., nanopore-based detection devices, lateral flow assay, smartphone-based detection devices, and microfluidic devices) is very promising for the on-site detection of agri-food contaminants. Additionally, CRISPR/Cas system-based biosensing technologies can be further integrated with much more innovative technologies for the development of novel detection platforms to realize the more reliable on-site detection of agri-food safety.

The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are promising molecular diagnostic tools for rapidly and precisely elucidating the structure and function of genomes due to their high specificity, programmability, and multi-system compatibility in nucleic acid recognition. Multiple parameters limit the ability of a CRISPR/Cas system to detect DNA or RNA. Consequently, it must be used in conjunction with other nucleic acid amplification techniques or signal detection techniques, and the reaction components and reaction conditions should be modified and optimized to maximize the detection performance of the CRISPR/Cas system against various targets. As the field continues to develop, CRISPR/Cas systems have the potential to become an ultra-sensitive, convenient, and accurate biosensing platform for the detection of specific target sequences. The design of a molecular detection platform employing the CRISPR/Cas system is asserted on three primary strategies: (1) Performance optimization of the CRISPR/Cas system; (2) enhancement of the detection signal and its interpretation; and (3) compatibility with multiple reaction systems. This article focuses on the molecular characteristics and application value of the CRISPR/Cas system and reviews recent research progress and development direction from the perspectives of principle, performance, and method development challenges to provide a theoretical foundation for the development and application of the CRISPR/CAS system in molecular detection technology.

Repurposing CRISPR/Cas to Discover SARS-CoV-2 Detecting and Neutralizing Aptamers.

RNA aptamers provide useful biological probes and therapeutic agents. New methodologies to screen RNA aptamers will be valuable by complementing the traditional Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Meanwhile, repurposing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated systems (Cas) has expanded their utility far beyond their native nuclease function. Here, CRISmers, a CRISPR/Cas-based novel screening system for RNA aptamers based on binding to a chosen protein of interest in a cellular context, is presented. Using CRISmers, aptamers are identified specifically targeting the receptor binding domain (RBD) of the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two aptamer leads enable sensitive detection and potent neutralization of SARS-CoV-2 Delta and Omicron variants in vitro. Intranasal administration of one aptamer, further modified with 2'-fluoro pyrimidines (2'-F), 2'-O-methyl purines (2'-O), and conjugation with both cholesterol and polyethylene glycol of 40 kDa (PEG40K), achieves effective prophylactic and therapeutic antiviral activity against live Omicron BA.2 variants in vivo. The study concludes by demonstrating the robustness, consistency, and potential broad utility of CRISmers using two newly identified aptamers but switching CRISPR, selection marker, and host species.

Optimized nanoparticle-mediated base editing of cystic fibrosis-causing variants

Background: Next-generation SARS-CoV-2 vaccines demonstrated that nanoparticle messenger ribonucleic acid (mRNA) delivery is effective and safe for in vivo delivery in humans. Current treatments for cystic fibrosis (CF) primarily focus on modulator drug therapies designed to correct malfunctioning CF transmembrane conductance regulator (CFTR) protein, but these modulators are ineffective for the 10% of people with CF with variants that do not allow protein production. Among these is the splice variant 3120 + 1G >A, the most common CF-causing mutation in native Africans. Gene editing would allow production of CFTR protein and enhancement of function using available CFTR modulators. We have demonstrated that electroporation of a modified CRISPR-Cas9 base editor to primary human bronchial epithelial cells carrying 3120 + 1G >A and F508del mutant alleles achieved 75% genome editing of the splice variant, resulting in approximately 40% wild-type (WT) CFTR function [1]. Here,we evaluate the effectiveness of several new nanoparticle formulations at delivering green fluorescent protein (GFP) mRNA to CF bronchial epithelial (CFBE41o-) cells. Using the optimal formulation,we then tested the efficacy correction of the 3120 + 1G >Avariant in a CFTR expression minigene (EMG) integrated into the genome of isogenic CFBE cells using mRNA and plasmid deoxyribonucleic acid (DNA) encoding adenine base editor (ABE) and guide (g)RNA. Method(s): GFP served as a reporter to evaluate transfection efficiency, cell viability, and mean fluorescence intensity (MFI) for three dosages (150, 75, 32.5 ng of mRNA), four polymer-to-mRNA to weight (w/w) ratios (60, 40, 30, 20), and four polymers (R, Y, G, B). 7-AAD served as a live/dead stain to quantify viability, with flow cytometry results analyzed using FlowJo software. CFBE cells stably expressing the 3120 + 1G >A EMG were transfected with the optimized nanoparticle formulation to deliver ABE and gRNA at two dosages (150, 75 ng) of mRNA and DNA. CFTR function in CFBE cellswas measured by short circuit current, forskolin stimulation, and inh-172 inhibition as a measure of editing efficiency. Result(s): Flow cytometry showed that polymer R achieved more than 85% GFP transfection, compared with a maximum of approximately 35% for the other three polymers at the maximum 150-ng dose, with approximately 80% viability normalized to untreated cells. In addition, polymer R achieved GFP MFI more than one order of magnitude as high as other formulations (~30 000 vs 2700 MFI) for the other three polymers at 150-ng dose and 40 w/w ratio. CFBE cells transfected with polymer R nanoparticles containing ABE and guide RNA at 75 ng and 150 ng showed mean CFTR function increase to 10 muA 6 (standard error of the mean [SEM] 1.1 muA) (~10% of WT) and 6.3 muA (SEM 0.9 muA) (~6% of WT), respectively. Greater toxicity at the higher dose could explain the larger increase in CFTR current at the lower dose. DNA-encoded ABE plasmid and gRNA showed a less robust increase in CFTR function (2.9 muA [SEM 0.4 muA] for 75-ng dose;3.0 muA [SEM 0.4 muA] for 150-ng dose), which was probably a result of the nanoparticle formulation being optimized for RNA instead of DNA cargo or the additional intracellular barriers that must be overcome for successful DNA delivery. Conclusion(s): We demonstrated that an optimized nanoparticle formulation containing ABE and gRNA can correct splicing of isogenic cells bearing the 3120 + 1G >A CFTR variant, resulting in recovery of CFTR function. In ongoing work, we are adapting these nanoparticles for RNA- and DNAencoded ABE and gRNA delivery to primary human bronchial epithelial cells.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Abstracts of the papers presented in the International Conference of Indian Virological Society, VIROCON 2022 on "Emerging and Re-emerging Viral Infections Impacting Humans, Animals, Plants, Fish and Environment"

The proceedings contain 206 papers. The topics discussed include: influenza: experiences from Kashmir;outbreaks of different viral etiologies amidst COVID-19 pandemic;development of a colorimetric isothermal (LAMP) assay for rapid detection of monkeypox virus;circulation of genetically diverse non-polio enteroviruses in respiratory samples during COVID-19 pandemic period (2021-22);evolutionary analysis of all eleven genes of species C rotaviruses circulating in humans and domestic animals;molecular characterization of dengue viruses circulating in Pune district, Maharashtra from 2009-2022;isolation and genomic characterization of cell fusing agent virus from aedes aegypti mosquitoes from Assam, India;structure-based identification and evaluation of antiviral activity of potent small molecule inhibitors targeting alphavirus RNA-dependent RNA polymerase;integration of HBV receptor NTCP into hepatoma cell using grnome editing;and hepatitis B virus genome targeting using CRISPR/Cas9based gene editing tool.

Four thermostatic steps: A novel CRISPR-Cas12-based system for the rapid at-home detection of respiratory pathogens.

The outbreak of coronavirus disease 2019 (COVID-19) in 2019 has severely damaged the world's economy and public health and made people pay more attention to respiratory infectious diseases. However, traditional quantitative real-time polymerase chain reaction (qRT-PCR) nucleic acid detection kits require RNA extraction, reverse transcription, and amplification, as well as the support of large-scale equipment to enrich and purify nucleic acids and precise temperature control. Therefore, novel, fast, convenient, sensitive and specific detection methods are urgently being developed and moving to proof of concept test. In this study, we developed a new nucleic acid detection system, referred to as 4 Thermostatic steps (4TS), which innovatively allows all the detection processes to be completed in a constant temperature device, which performs extraction, amplification, cutting of targets, and detection within 40 min. The assay can specifically and sensitively detect five respiratory pathogens, namely SARS-CoV-2, Mycoplasma felis (MF), Chlamydia felis (CF), Feline calicivirus (FCV), and Feline herpes virus (FHV). In addition, a cost-effective and practical small-scale reaction device was designed and developed to maintain stable reaction conditions. The results of the detection of the five viruses show that the sensitivity of the system is greater than 94%, and specificity is 100%. The 4TS system does not require complex equipment, which makes it convenient and fast to operate, and allows immediate testing for suspected infectious agents at home or in small clinics. Therefore, the assay system has diagnostic value and significant potential for further reducing the cost of early screening of infectious diseases and expanding its application. KEY POINTS: • The 4TS system enables the accurate and specific detection of nucleic acid of pathogens at 37 °C in four simple steps, and the whole process only takes 40 min. •A simple alkali solution can be used to extract nucleic acid. • A small portable device simple to operate is developed for home diagnosis and detection of respiratory pathogens.

Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.

CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.