Inverse regulation of SOS1 and HKT1 protein localization and stability by SOS3/CBL4 in Arabidopsis thaliana.

To control net sodium (Na+) uptake, Arabidopsis plants utilize the plasma membrane (PM) Na+/H+ antiporter SOS1 to achieve Na+ efflux at the root and Na+ loading into the xylem, and the channel-like HKT1;1 protein that mediates the reverse flux of Na+ unloading off the xylem. Together, these opposing transport systems govern the partition of Na+ within the plant yet they must be finely co-regulated to prevent a futile cycle of xylem loading and unloading. Here, we show that the Arabidopsis SOS3 protein acts as the molecular switch governing these Na+ fluxes by favoring the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 kinase complex under salt stress, while commanding HKT1;1 protein degradation upon acute sodic stress. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1. These results indicate that roots first retain moderate amounts of salts to facilitate osmoregulation, yet when sodicity exceeds a set point, SOS3-dependent HKT1;1 degradation switches the balance toward Na+ export out of the root. Thus, SOS3 functionally links and co-regulates the two major Na+ transport systems operating in vascular plants controlling plant tolerance to salinity.

Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2.

Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/µL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

Molecular farming for sustainable production of clinical-grade antimicrobial peptides.

Antimicrobial peptides (AMPs) are emerging as next-generation therapeutics due to their broad-spectrum activity against drug-resistant bacterial strains and their ability to eradicate biofilms, modulate immune responses, exert anti-inflammatory effects and improve disease management. They are produced through solid-phase peptide synthesis or in bacterial or yeast cells. Molecular farming, i.e. the production of biologics in plants, offers a low-cost, non-toxic, scalable and simple alternative platform to produce AMPs at a sustainable cost. In this review, we discuss the advantages of molecular farming for producing clinical-grade AMPs, advances in expression and purification systems and the cost advantage for industrial-scale production. We further review how 'green' production is filling the sustainability gap, streamlining patent and regulatory approvals and enabling successful clinical translations that demonstrate the future potential of AMPs produced by molecular farming. Finally, we discuss the regulatory challenges that need to be addressed to fully realize the potential of molecular farming-based AMP production for therapeutics.

PNA-Pdx: Versatile Peptide Nucleic Acid-Based Detection of Nucleic Acids and SNPs.

Monitoring diseases caused by pathogens or by mutations in DNA sequences requires accurate, rapid, and sensitive tools to detect specific nucleic acid sequences. Here, we describe a new peptide nucleic acid (PNA)-based nucleic acid detection toolkit, termed PNA-powered diagnostics (PNA-Pdx). PNA-Pdx employs PNA probes that bind specifically to a target and are then detected in lateral flow assays. This can precisely detect a specific pathogen or genotype genomic sequence. PNA probes can also be designed to invade double-stranded DNAs (dsDNAs) to produce single-stranded DNAs for precise CRISPR-Cas12b-based detection of genomic SNPs without requiring the protospacer-adjacent motif (PAM), as Cas12b requires PAM sequences only for dsDNA targets. PNA-Pdx identified target nucleic acid sequences at concentrations as low as 2 copies/µL and precisely detected the SARS-CoV-2 genome in clinical samples in 40 min. Furthermore, the specific dsDNA invasion by the PNA coupled with CRISPR-Cas12b precisely detected genomic SNPs without PAM restriction. Overall, PNA-Pdx provides a novel toolkit for nucleic acid and SNP detection as well as highlights the benefits of engineering PNA probes for detecting nucleic acids.

A CRISPR-based lateral flow assay for plant genotyping and pathogen diagnostics.

Efficient pathogen diagnostics and genotyping methods enable effective disease management and breeding, improve crop productivity and ensure food security. However, current germplasm selection and pathogen detection techniques are laborious, time-consuming, expensive and not easy to mass-scale application in the field. Here, we optimized a field-deployable lateral flow assay, Bio-SCAN, as a highly sensitive tool to precisely identify elite germplasm and detect mutations, transgenes and phytopathogens in <1 h, starting from sample isolation to result output using lateral flow strips. As a proof of concept, we genotyped various wheat germplasms for the Lr34 and Lr67 alleles conferring broad-spectrum resistance to stripe rust, confirmed the presence of synthetically produced herbicide-resistant alleles in the rice genome and screened for the presence of transgenic elements in the genome of transgenic tobacco and rice plants with 100% specificity. We also successfully applied this new assay to the detection of phytopathogens, including viruses and bacterial pathogens in Nicotiana benthamiana, and two destructive fungal pathogens (Puccinia striiformis f. sp. tritici and Magnaporthe oryzae Triticum) in wheat. Our results illustrate the power of Bio-SCAN in crop breeding, genetic engineering and pathogen diagnostics to enhance food security. The high sensitivity, simplicity, versatility and in-field deployability make the Bio-SCAN as an attractive molecular diagnostic tool for diverse applications in agriculture.

CRISPR/Cas systems versus plant viruses: engineering plant immunity and beyond.

Molecular engineering of plant immunity to confer resistance against plant viruses holds great promise for mitigating crop losses and improving plant productivity and yields, thereby enhancing food security. Several approaches have been employed to boost immunity in plants by interfering with the transmission or lifecycles of viruses. In this review, we discuss the successful application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (CRISPR/Cas) systems to engineer plant immunity, increase plant resistance to viruses, and develop viral diagnostic tools. Furthermore, we examine the use of plant viruses as delivery systems to engineer virus resistance in plants and provide insight into the limitations of current CRISPR/Cas approaches and the potential of newly discovered CRISPR/Cas systems to engineer better immunity and develop better diagnostics tools for plant viruses. Finally, we outline potential solutions to key challenges in the field to enable the practical use of these systems for crop protection and viral diagnostics.

Vigilant: An Engineered VirD2-Cas9 Complex for Lateral Flow Assay-Based Detection of SARS-CoV2.

Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant (VirD2-dCas9 guided and LFA-coupled nucleic acid test), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/µL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.

A mutation in the major autophagy gene, WIPI2, associated with global developmental abnormalities.

We describe a large consanguineous pedigree from a remote area of Northern Pakistan, with a complex developmental disorder associated with wide-ranging symptoms, including mental retardation, speech and language impairment and other neurological, psychiatric, skeletal and cardiac abnormalities. We initially carried out a genetic study using the HumanCytoSNP-12 v2.1 Illumina gene chip on nine family members and identified a single region of homozygosity shared amongst four affected individuals on chromosome 7p22 (positions 3059377-5478971). We performed whole-exome sequencing on two affected individuals from two separate branches of the extended pedigree and identified a novel nonsynonymous homozygous mutation in exon 9 of the WIPI2 (WD-repeat protein interacting with phosphoinositide 2) gene at position 5265458 (c.G745A;pV249M). WIPI2 plays a critical role in autophagy, an evolutionary conserved cellular pathway implicated in a growing number of medical conditions. The mutation is situated in a highly conserved and critically important region of WIPI2, responsible for binding PI(3)P and PI(3,5)P2, an essential requirement for autophagy to proceed. The mutation is absent in all public databases, is predicted to be damaging and segregates with the disease phenotype. We performed functional studies in vitro to determine the potential effects of the mutation on downstream pathways leading to autophagosome assembly. Binding of the V231M mutant of WIPI2b to ATG16L1 (as well as ATG5-12) is significantly reduced in GFP pull-down experiments, and fibroblasts derived from the patients show reduced WIPI2 puncta, reduced LC3 lipidation and reduced autophagic flux.

High efficiency of targeted mutagenesis in arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease.

KEY MESSAGE: The use of a meiosis I-specific promoter increased the efficiency of targeted mutagenesis and will facilitate the manipulation of homologous recombination. The CRISPR/Cas9 system has been harnessed for targeted engineering of eukaryotic genomes, including plants; however, CRISPR/Cas9 efficiency varies considerably in different plant tissues and species. In Arabidopsis, the generation of homozygous or bi-allelic mutants in the first (T1) generation is inefficient. Here, we used specific promoters to drive the expression of Cas9 during meiosis to maximize the efficiency of recovering heritable mutants in T1 plants. Our data reveal that the use of a promoter active in meiosis I resulted in high-efficiency (28 %) recovery of targeted mutants in the T1 generation. Moreover, this method enabled efficient simultaneous targeting of three genes for mutagenesis. Taken together, our results show that the use of meiosis-specific promoters will improve methods for functional genomic analysis and studying the molecular underpinnings of homologous recombination.

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.

Pharmacoeconomic analysis of palifermin to prevent mucositis among patients undergoing autologous hematopoietic stem cell transplantation.

Trials have shown benefits of palifermin in reducing the incidence and severity of oral mucositis in patients with hematological malignancies undergoing autologous hematopoietic stem cell transplantation (HSCT) with total body irradiation (TBI)-based conditioning regimens. Similar outcome data are lacking for patients receiving non-TBI-based regimens. We performed a retrospective evaluation on the pharmacoeconomic benefit of palifermin in the setting of non-TBI-based conditioning and autologous HSCT. Between January 2002 and December 2010, 524 patients undergoing autologous HSCT for myeloma (melphalan 200 mg/m²) and lymphoma (high-dose busulfan, cyclophosphamide, and etoposide) as preparative regimen were analyzed. Use of patient-controlled analgesia (PCA) was significantly lower in the palifermin-treated groups (myeloma: 13% versus 53%, P < .001; lymphoma: 46% versus 68%, P < .001). Median total transplant charges were significantly higher in the palifermin-treated group, after controlling for inflation (myeloma: $167,820 versus $143,200, P < .001; lymphoma: $168,570 versus $148,590, P < .001). Palifermin treatment was not associated with a difference in days to neutrophil engraftment, length of stay, and overall survival and was associated with an additional cost of $5.5K (myeloma) and $14K (lymphoma) per day of PCA avoided. Future studies are suggested to evaluate the cost-effectiveness of palifermin compared with other symptomatic treatments to reduce transplant toxicity using validated measures for pain and quality of life.

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.

TsHKT1;2, a HKT1 homolog from the extremophile Arabidopsis relative Thellungiella salsuginea, shows K(+) specificity in the presence of NaCl.

Cellular Na(+)/K(+) ratio is a crucial parameter determining plant salinity stress resistance. We tested the function of plasma membrane Na(+)/K(+) cotransporters in the High-affinity K(+) Transporter (HKT) family from the halophytic Arabidopsis (Arabidopsis thaliana) relative Thellungiella salsuginea. T. salsuginea contains at least two HKT genes. TsHKT1;1 is expressed at very low levels, while the abundant TsHKT1;2 is transcriptionally strongly up-regulated by salt stress. TsHKT-based RNA interference in T. salsuginea resulted in Na(+) sensitivity and K(+) deficiency. The athkt1 mutant lines overexpressing TsHKT1;2 proved less sensitive to Na(+) and showed less K(+) deficiency than lines overexpressing AtHKT1. TsHKT1;2 ectopically expressed in yeast mutants lacking Na(+) or K(+) transporters revealed strong K(+) transporter activity and selectivity for K(+) over Na(+). Altering two amino acid residues in TsHKT1;2 to mimic the AtHKT1 sequence resulted in enhanced sodium uptake and loss of the TsHKT1;2 intrinsic K(+) transporter activity. We consider the maintenance of K(+) uptake through TsHKT1;2 under salt stress an important component supporting the halophytic lifestyle of T. salsuginea.

Bio-SCAN V2: A CRISPR/dCas9-based lateral flow assay for rapid detection of theophylline.

Rapid, specific, and robust diagnostic strategies are needed to develop sensitive biosensors for small molecule detection, which could aid in controlling contamination and disease transmission. Recently, the target-induced collateral activity of Cas nucleases [clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases] was exploited to develop high-throughput diagnostic modules for detecting nucleic acids and small molecules. Here, we have expanded the diagnostic ability of the CRISPR-Cas system by developing Bio-SCAN V2, a ligand-responsive CRISPR-Cas platform for detecting non-nucleic acid small molecule targets. The Bio-SCAN V2 consists of an engineered ligand-responsive sgRNA (ligRNA), biotinylated dead Cas9 (dCas9-biotin), 6-carboxyfluorescein (FAM)-labeled amplicons, and lateral flow assay (LFA) strips. LigRNA interacts with dCas9-biotin only in the presence of sgRNA-specific ligand molecules to make a ribonucleoprotein (RNP). Next, the ligand-induced ribonucleoprotein is exposed to FAM-labeled amplicons for binding, and the presence of the ligand (small molecule) is detected as a visual signal [(dCas9-biotin)-ligRNA-FAM labeled DNA-AuNP complex] at the test line of the lateral flow assay strip. With the Bio-SCAN V2 platform, we are able to detect the model molecule theophylline with a limit of detection (LOD) up to 2 µM in a short time, requiring only 15 min from sample application to visual readout. Taken together, Bio-SCAN V2 assay provides a rapid, specific, and ultrasensitive detection platform for theophylline.

Improvement of Soybean; A Way Forward Transition from Genetic Engineering to New Plant Breeding Technologies.

Soybean is considered one of the important crops among legumes. Due to high nutritional contents in seed (proteins, sugars, oil, fatty acids, and amino acids), soybean is used globally for food, feed, and fuel. The primary consumption of soybean is vegetable oil and feed for chickens and livestock. Apart from this, soybean benefits soil fertility by fixing atmospheric nitrogen through root nodular bacteria. While conventional breeding is practiced for soybean improvement, with the advent of new biotechnological methods scientists have also engineered soybean to improve different traits (herbicide, insect, and disease resistance) to fulfill consumer requirements and to meet the global food deficiency. Genetic engineering (GE) techniques such as transgenesis and gene silencing help to minimize the risks and increase the adaptability of soybean. Recently, new plant breeding technologies (NPBTs) emerged such as zinc-finger nucleases, transcription activator-like effector nucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9), which paved the way for enhanced genetic modification of soybean. These NPBTs have the potential to improve soybean via gene functional characterization precision genome engineering for trait improvement. Importantly, these NPBTs address the ethical and public acceptance issues related to genetic modifications and transgenesis in soybean. In the present review, we summarized the improvement of soybean through GE and NPBTs. The valuable traits that have been improved through GE for different constraints have been discussed. Moreover, the traits that have been improved through NPBTs and potential targets for soybean improvements via NPBTs and solutions for ethical and public acceptance are also presented.

Efficient in planta production of amidated antimicrobial peptides that are active against drug-resistant ESKAPE pathogens.

Antimicrobial peptides (AMPs) are promising next-generation antibiotics that can be used to combat drug-resistant pathogens. However, the high cost involved in AMP synthesis and their short plasma half-life render their clinical translation a challenge. To address these shortcomings, we report efficient production of bioactive amidated AMPs by transient expression of glycine-extended AMPs in Nicotiana benthamiana line expressing the mammalian enzyme peptidylglycine α-amidating mono-oxygenase (PAM). Cationic AMPs accumulate to substantial levels in PAM transgenic plants compare to nontransgenic N. benthamiana. Moreover, AMPs purified from plants exhibit robust killing activity against six highly virulent and antibiotic resistant ESKAPE pathogens, prevent their biofilm formation, analogous to their synthetic counterparts and synergize with antibiotics. We also perform a base case techno-economic analysis of our platform, demonstrating the potential economic advantages and scalability for industrial use. Taken together, our experimental data and techno-economic analysis demonstrate the potential use of plant chassis for large-scale production of clinical-grade AMPs.

Pharmacokinetic-directed high-dose busulfan combined with cyclophosphamide and etoposide results in predictable drug levels and durable long-term survival in lymphoma patients undergoing autologous stem cell transplantation.

The clinical advantage of pharmacokinetic (PK)-directed-based dosing on intravenous (i.v.) versus oral busulfan-related toxicity and survival remains unclear. We performed a retrospective cohort study of sequential cohorts of patients comparing PK-directed oral and i.v. busulfan-based conditioning regimens in lymphoma patients undergoing autologous hematopoietic cell transplantation (ASCT). Patients received oral (n = 95), every 6 hours i.v. (IV16, n = 113), or once-daily i.v. (IV4, n = 86) busulfan, cyclophosphamide, and etoposide. PK-directed dosing was performed to achieve a predefined target area under the curve (AUC) of 20,000 µM-min (range: 18,400-21,600 µM-min). PK-directed dose adjustments markedly reduced the number of patients in the oral group with total AUC higher than the targeted AUC range, and reduced the variations of total AUC values in all patient groups. One hundred-day mortality was 2.1%, 3.6%, and 3.5% for oral, IV16, and IV4 cohorts, respectively. Five-year overall survival (OS) was 57% (95% confidence interval [CI] 45%-66%) and 64% (95% CI 53%-73%) for patients who received oral and i.v. busulfan, respectively. Both multivariable and instrumental variable analyses indicated the route of delivery had no significant impact on OS, whereas refractory disease and age ≥55 were significantly associated with poorer OS. In lymphoma patients undergoing ASCT, PK-directed i.v. or oral busulfan-based conditioning regimens have comparable toxicity and OS.

CS-Cells: A CRISPR-Cas12 DNA Device to Generate Chromosome-Shredded Cells for Efficient and Safe Molecular Biomanufacturing.

Synthetic biology holds great promise for translating ideas into products to address the grand challenges facing humanity. Molecular biomanufacturing is an emerging technology that facilitates the production of key products of value, including therapeutics and select chemical compounds. Current biomanufacturing technologies require improvements to overcome limiting factors, including efficient production, cost, and safe release; therefore, developing optimum chassis for biomolecular manufacturing is of great interest for enabling diverse synthetic biology applications. Here, we harnessed the power of the CRISPR-Cas12 system to design, build, and test a DNA device for genome shredding, which fragments the native genome to enable the conversion of bacterial cells into nonreplicative, biosynthetically active, and programmable molecular biomanufacturing chassis. As a proof of concept, we demonstrated the efficient production of green fluorescent protein and violacein, an antimicrobial and antitumorigenic compound. Our CRISPR-Cas12-based chromosome-shredder DNA device has built-in biocontainment features providing a roadmap for the conversion of any bacterial cell into a chromosome-shredded chassis amenable to high-efficiency molecular biomanufacturing, thereby enabling exciting and diverse biotechnological applications.

Twin prime editor: seamless repair without damage.

CRISPR-Cas9 creates remarkable possibilities to modify targeted regions in genomic DNA. However, CRISPR-Cas-mediated DNA double-stranded breaks (DSBs), that tend to generate random insertions or deletions, limit this technology. Recently, Anzalone et al. developed a 'twin prime editing' tool to replace, integrate, or delete large genomic DNA sequences without generating DNA DSBs.

Bio-SCAN: A CRISPR/dCas9-Based Lateral Flow Assay for Rapid, Specific, and Sensitive Detection of SARS-CoV-2.

Simple, rapid, specific, and sensitive point-of-care detection methods are needed to contain the spread of SARS-CoV-2. CRISPR/Cas9-based lateral flow assays are emerging as a powerful alternative for COVID-19 diagnostics. Here, we developed Bio-SCAN (biotin-coupled specific CRISPR-based assay for nucleic acid detection) as an accurate pathogen detection platform that requires no sophisticated equipment or technical expertise. Bio-SCAN detects the SARS-CoV-2 genome in less than 1 h from sample collection to result. In the first step, the target nucleic acid sequence is isothermally amplified in 15 min via recombinase polymerase amplification before being precisely detected by biotin-labeled nuclease-dead SpCas9 (dCas9) on commercially available lateral flow strips. The resulting readout is visible to the naked eye. Compared to other CRISPR-Cas-based pathogen detection assays, Bio-SCAN requires no additional reporters, probes, enhancers, reagents, or sophisticated devices to interpret the results. Bio-SCAN is highly sensitive and successfully detected a clinically relevant level (4 copies/µL) of synthetic SARS-CoV-2 RNA genome. Similarly, Bio-SCAN showed 100% negative and 96% positive predictive agreement with RT-qPCR results when using clinical samples (86 nasopharyngeal swab samples). Furthermore, incorporating variant-specific sgRNAs in the detection reaction allowed Bio-SCAN to efficiently distinguish between the α, ß, and δ SARS-CoV-2 variants. Also, our results confirmed that the Bio-SCAN reagents have a long shelf life and can be assembled locally in nonlaboratory and limited-resource settings. Furthermore, the Bio-SCAN platform is compatible with the nucleic acid quick extraction protocol. Our results highlight the potential of Bio-SCAN as a promising point-of-care diagnostic platform that can facilitate low-cost mass screening for SARS-CoV-2.