Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks.

BACKGROUND: CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks. RESULTS: Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. CONCLUSIONS: Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

Transcriptomic and metabolomic changes in postharvest sugarbeet roots reveal widespread metabolic changes in storage and identify genes potentially responsible for respiratory sucrose loss.

Endogenous metabolism is primarily responsible for losses in sucrose content and processing quality in postharvest sugarbeet roots. The genes responsible for this metabolism and the transcriptional changes that regulate it, however, are largely unknown. To identify genes and metabolic pathways that participate in postharvest sugarbeet root metabolism and the transcriptional changes that contribute to their regulation, transcriptomic and metabolomic profiles were generated for sugarbeet roots at harvest and after 12, 40 and 120 d storage at 5 and 12°C and gene expression and metabolite concentration changes related to storage duration or temperature were identified. During storage, 8656 genes, or 34% of all expressed genes, and 225 metabolites, equivalent to 59% of detected metabolites, were altered in expression or concentration, indicating extensive transcriptional and metabolic changes in stored roots. These genes and metabolites contributed to a wide range of cellular and molecular functions, with carbohydrate metabolism being the function to which the greatest number of genes and metabolites classified. Because respiration has a central role in postharvest metabolism and is largely responsible for sucrose loss in sugarbeet roots, genes and metabolites involved in and correlated to respiration were identified. Seventy-five genes participating in respiration were differentially expressed during storage, including two bidirectional sugar transporter SWEET17 genes that highly correlated with respiration rate. Weighted gene co-expression network analysis identified 1896 additional genes that positively correlated with respiration rate and predicted a pyruvate kinase gene to be a central regulator or biomarker for respiration rate. Overall, these results reveal the extensive and diverse physiological and metabolic changes that occur in stored sugarbeet roots and identify genes with potential roles as regulators or biomarkers for respiratory sucrose loss.

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public's health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

An open-source, automated, and cost-effective platform for COVID-19 diagnosis and rapid portable genomic surveillance using nanopore sequencing.

The COVID-19 pandemic, caused by SARS-CoV-2, has emphasized the necessity for scalable diagnostic workflows using locally produced reagents and basic laboratory equipment with minimal dependence on global supply chains. We introduce an open-source automated platform for high-throughput RNA extraction and pathogen diagnosis, which uses reagents almost entirely produced in-house. This platform integrates our methods for self-manufacturing magnetic nanoparticles and qRT-PCR reagents-both of which have received regulatory approval for clinical use-with an in-house, open-source robotic extraction protocol. It also incorporates our "Nanopore Sequencing of Isothermal Rapid Viral Amplification for Near Real-time Analysis" (NIRVANA) technology, designed for tracking SARS-CoV-2 mutations and variants. The platform exhibits high reproducibility and consistency without cross-contamination, and its limit of detection, sensitivity, and specificity are comparable to commercial assays. Automated NIRVANA effectively identifies circulating SARS-CoV-2 variants. Our in-house, cost-effective reagents, automated diagnostic workflows, and portable genomic surveillance strategies provide a scalable and rapid solution for COVID-19 diagnosis and variant tracking, essential for current and future pandemic responses.

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.

Interaction of human HelQ with DNA polymerase delta halts DNA synthesis and stimulates DNA single-strand annealing.

DNA strand breaks are repaired by DNA synthesis from an exposed DNA end paired with a homologous DNA template. DNA polymerase delta (Pol δ) catalyses DNA synthesis in multiple eukaryotic DNA break repair pathways but triggers genome instability unless its activity is restrained. We show that human HelQ halts DNA synthesis by isolated Pol δ and Pol δ-PCNA-RPA holoenzyme. Using novel HelQ mutant proteins we identify that inhibition of Pol δ is independent of DNA binding, and maps to a 70 amino acid intrinsically disordered region of HelQ. Pol δ and its POLD3 subunit robustly stimulated DNA single-strand annealing by HelQ, and POLD3 and HelQ interact physically via the intrinsically disordered HelQ region. This data, and inability of HelQ to inhibit DNA synthesis by the POLD1 catalytic subunit of Pol δ, reveal a mechanism for limiting DNA synthesis and promoting DNA strand annealing during human DNA break repair, which centres on POLD3.

A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repair.

Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.

Nucleotide excision repair (NER) removes bulky DNA lesions and is thereby crucial in maintaining transcription and genomic integrity. Here, the authors show a dual function for the XPG nuclease that is critical for finding and excising the damage. During the separation of the damage-containing strand from the undamaged strand, XPG stimulates TFIIH dependent dsDNA unwinding 10 fold. In return, when TFIIH stalls at the damage it stimulates XPG nuclease activity 700 fold. Remarkably, this mutually exclusive coordination requires a bubble longer than 15 nucleotides. This study addressees why a bubble of a certain size is needed to facilitate NER and why XPG is recruited at the beginning of NER when its endonucleolytic activity is required at the very end.

Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing.

During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics.

The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation. Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer, with an intermediary nick structure generated for each cycle. Here, we show that human Polδ is inefficient in releasing the nick product from FEN1, resulting in non-processive and remarkably slow RNA removal. Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation. These mechanisms are coordinated by PCNA, which encircles DNA, and dynamically recruits Polδ, FEN1, and Lig1 to compete for their substrates. Our findings call for investigating additional pathways that may accelerate RNA removal in human cells, such as RNA pre-removal by RNase Hs, which, as demonstrated herein, enhances the maturation rate ~10-fold. They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination.

Cryo-electron structures of the extreme thermostable enzymes Sulfur Oxygenase Reductase and Lumazine Synthase.

Thermostable enzymes have the potential for use in a wide variety of biotechnological applications. Cryo-electron microscopy (cryo-EM) enables the imaging of biomolecules in their native aqueous environment. Here, we present high resolution cryo-EM structures of two thermostable enzymes that exhibit multimeric cage-like structures arranged into two different point-group symmetries. First, we determined the structure of the Sulfur Oxygenase Reductase (SOR) enzyme that catalyzes both the oxygenation and disproportionation of elemental sulfur in Archea and is composed of 24 homomeric units each of MW ≃ 35 kDa arranged in octahedral symmetry. The structure of SOR from Acidianus ambivalens (7X9W) was determined at 2.78 Å resolution. The active site of each subunit inside the central nanocompartment is composed of Fe3+ coordinated to two water molecules and the three amino acids (H86, H90 and E114). Second, we determined the structure of Lumazine Synthase (LS) from Aquifex aeolicus (7X7M) at 2.33 Å resolution. LS forms a cage-like structure consisting of 60 identical subunits each of MW ≃ 15 kDa arranged in a strict icosahedral symmetry. The LS subunits are interconnected by ion-pair network. Due to their thermostability and relatively easy purification scheme, both SOR and LS can serve as a model for the catalytic and structural characterization of biocatalysts as well as a benchmark for cryo-EM sample preparation, optimization of the acquisition parameters and 3D reconstruction.

Exploring the Genetic Diversity and Population Structure of Wheat Landrace Population Conserved at ICARDA Genebank.

Landraces are considered a valuable source of potential genetic diversity that could be used in the selection process in any plant breeding program. Here, we assembled a population of 600 bread wheat landraces collected from eight different countries, conserved at the ICARDA's genebank, and evaluated the genetic diversity and the population structure of the landraces using single nucleotide polymorphism (SNP) markers. A total of 11,830 high-quality SNPs distributed across the genomes A (40.5%), B (45.9%), and D (13.6%) were used for the final analysis. The population structure analysis was evaluated using the model-based method (STRUCTURE) and distance-based methods [discriminant analysis of principal components (DAPC) and principal component analysis (PCA)]. The STRUCTURE method grouped the landraces into two major clusters, with the landraces from Syria and Turkey forming two clusters with high proportions of admixture, whereas the DAPC and PCA analysis grouped the population into three subpopulations mostly according to the geographical information of the landraces, i.e., Syria, Iran, and Turkey with admixture. The analysis of molecular variance revealed that the majority of the variation was due to genetic differences within the populations as compared with between subpopulations, and it was the same for both the cluster-based and distance-based methods. Genetic distance analysis was also studied to estimate the differences between the landraces from different countries, and it was observed that the maximum genetic distance (0.389) was between the landraces from Spain and Palestine, whereas the minimum genetic distance (0.013) was observed between the landraces from Syria and Turkey. It was concluded from the study that the model-based methods (DAPC and PCA) could dissect the population structure more precisely when compared with the STRUCTURE method. The population structure and genetic diversity analysis of the bread wheat landraces presented here highlight the complex genetic architecture of the landraces native to the Fertile Crescent region. The results of this study provide useful information for the genetic improvement of hexaploid wheat and facilitate the use of landraces in wheat breeding programs.

QTL Mapping of Adult Plant Resistance to Stripe Rust in a Doubled Haploid Wheat Population.

Stripe rust caused by Puccinia striiformis Westend. f. sp. tritici. is a major bread wheat disease worldwide with yield losses of up to 100% under severe disease pressure. The deployment of resistant cultivars with adult plant resistance to the disease provides a long-term solution to stripe rust of wheat. An advanced line from the International Winter Wheat Improvement Program (IWWIP) 130675 (Avd/Vee#1//1-27-6275/Cf 1770/3/MV171-C-17466) showed a high level of adult plant resistance to stripe rust in the field. To identify the adult plant resistance genes in this elite line, a mapping population of 190 doubled haploid (DH) lines was developed from a cross between line 130675 and the universal stripe rust-susceptible variety Avocet S. The DH population was evaluated at precision wheat stripe rust phenotyping platform, in Izmir during 2019, 2020, and 2021 cropping seasons under artificial inoculations. Composite interval mapping (CIM) identified two stable QTLs QYr.rcrrc-3B.1, and QYr.rcrrc-3B.2, which were detected in multiple years. In addition to these two QTLs, five more QTLs, QYr.rcrrc-1B, QYr.rcrrc-2A, QYr.rcrrc-3A, QYr.rcrrc-5A, and QYr.rcrrc-7D, were identified, which were specific to the cropping year (environment). All QTLs were derived from the resistant parent, except QYr.rcrrc-3A. The significant QTLs explained 3.4-20.6% of the phenotypic variance. SNP markers flanking the QTL regions can be amenable to marker-assisted selection. The best DH lines with high yield, end-use quality, and stripe rust resistance can be used for further selection for improved germplasm. SNP markers flanking the QTL regions can aid in identifying such lines.

Wiskott-Aldrich syndrome protein forms nuclear condensates and regulates alternative splicing.

The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.

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.

Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA.

Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.

Implementing fluorescence enhancement, quenching, and FRET for investigating flap endonuclease 1 enzymatic reaction at the single-molecule level.

Flap endonuclease 1 (FEN1) is an important component of the intricate molecular machinery for DNA replication and repair. FEN1 is a structure-specific 5' nuclease that cleaves nascent single-stranded 5' flaps during the maturation of Okazaki fragments. Here, we review our research primarily applying single-molecule fluorescence to resolve important mechanistic aspects of human FEN1 enzymatic reaction. The methodology presented in this review is aimed as a guide for tackling other biomolecular enzymatic reactions by fluorescence enhancement, quenching, and FRET and their combinations. Using these methods, we followed in real-time the structures of the substrate and product and 5' flap cleavage during catalysis. We illustrate that FEN1 actively bends the substrate to verify its features and continues to mold it to induce a protein disorder-to-order transitioning that controls active site assembly. This mechanism suppresses off-target cleavage of non-cognate substrates and promotes their dissociation with an accuracy that was underestimated from bulk assays. We determined that product release in FEN1 after the 5' flap release occurs in two steps; a brief binding to the bent nicked-product followed by longer binding to the unbent nicked-product before dissociation. Based on our cryo-electron microscopy structure of the human lagging strand replicase bound to FEN1, we propose how this two-step product release mechanism may regulate the final steps during the maturation of Okazaki fragments.

Comparison of Genomic Prediction Methods for Yellow, Stem, and Leaf Rust Resistance in Wheat Landraces from Afghanistan.

Wheat rust diseases, including yellow rust (Yr; also known as stripe rust) caused by Puccinia striiformis Westend. f. sp. tritici, leaf rust (Lr) caused by Puccinia triticina Eriks. and stem rust (Sr) caused by Puccinia graminis Pres f. sp. tritici are major threats to wheat production all around the globe. Durable resistance to wheat rust diseases can be achieved through genomic-assisted prediction of resistant accessions to increase genetic gain per unit time. Genomic prediction (GP) is a promising technology that uses genomic markers to estimate genomic-assisted breeding values (GBEVs) for selecting resistant plant genotypes and accumulating favorable alleles for adult plant resistance (APR) to wheat rust diseases. To evaluate GP we compared the predictive ability of nine different parametric, semi-parametric and Bayesian models including Genomic Unbiased Linear Prediction (GBLUP), Ridge Regression (RR), Least Absolute Shrinkage and Selection Operator (LASSO), Elastic Net (EN), Bayesian Ridge Regression (BRR), Bayesian A (BA), Bayesian B (BB), Bayesian C (BC) and Reproducing Kernel Hilbert Spacing model (RKHS) to estimate GEBV's for APR to yellow, leaf and stem rust of wheat in a panel of 363 bread wheat landraces of Afghanistan origin. Based on five-fold cross validation the mean predictive abilities were 0.33, 0.30, 0.38, and 0.33 for Yr (2016), Yr (2017), Lr, and Sr, respectively. No single model outperformed the rest of the models for all traits. LASSO and EN showed the lowest predictive ability in four of the five traits. GBLUP and RR gave similar predictive abilities, whereas Bayesian models were not significantly different from each other as well. We also investigated the effect of the number of genotypes and the markers used in the analysis on the predictive ability of the GP model. The predictive ability was highest with 1000 markers and there was a linear trend in the predictive ability and the size of the training population. The results of the study are encouraging, confirming the feasibility of GP to be effectively applied in breeding programs for resistance to all three wheat rust diseases.

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.