Target DNA Research Articles

Programmable RNA detection with CRISPR-Cas12a

Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.

HLA Class I Expression Is Associated with DNA Damage and Immune Cell Infiltration into Dysplastic and Neoplastic Lesions in Ulcerative Colitis.

Human leukocyte antigen class I (HLA-I) is considered a genetic pathogen for ulcerative colitis (UC). This study aimed to investigate the significance of DNA damage and HLA-I expression in infiltrating immune cells and immune checkpoint protein PD-L1 expression in dysplasia/colitic cancer (CC) and sporadic colorectal cancer (SCRC). We performed immunohistochemical staining for HLA-I, PD-L1, γH2AX (DNA damage marker), and immune cell markers such as CD8, FOXP3, CD68, and CD163 (in surgically resected specimens from 17 SCRC patients with 12 adjacent normal mucosa (NM) and 9 UC patients with 18 dysplasia/CC tumors. The ratio of membrane HLA-I-positive epithelial cells in UC and dysplasia/CC tissues was significantly higher than that in NM and SCRC. High HLA-I expression in dysplasia/CC was associated with high positivity of γH2AX and PD-L1 expression compared to SCRC. The infiltration of CD8-positive T cells and CD68-positive macrophages in HLA-I-high dysplasia/CC was significantly higher than in UC and SCRC. Dysplasia/CC specimens with DNA damage exhibited high levels of HLA-I-positive epithelial cells with high CD8- and CD68-positive immune cell infiltration compared to UC and SCRC specimens. Targeting DNA damage in UC may regulate immune cell infiltration, immune checkpoint proteins, and carcinogenesis by modulating DNA damage-induced HLA-I antigen presentation.

The structure of AcrIC9 revealing the putative inhibitory mechanism of AcrIC9 against the type IC CRISPR-Cas system.

CRISPR-Cas systems are known to be part of the bacterial adaptive immune system that provides resistance against intruders such as viruses, phages and other mobile genetic elements. To combat this bacterial defense mechanism, phages encode inhibitors called Acrs (anti-CRISPR proteins) that can suppress them. AcrIC9 is the most recently identified member of the AcrIC family that inhibits the type IC CRISPR-Cas system. Here, the crystal structure of AcrIC9 from Rhodobacter capsulatus is reported, which comprises a novel fold made of three central antiparallel β-strands surrounded by three α-helixes, a structure that has not been detected before. It is also shown that AcrIC9 can form a dimer via disulfide bonds generated by the Cys69 residue. Finally, it is revealed that AcrIC9 directly binds to the type IC cascade. Analysis and comparison of its structure with structural homologs indicate that AcrIC9 belongs to DNA-mimic Acrs that directly bind to the cascade complex and hinder the target DNA from binding to the cascade.

Rapid Multiplex PCR for Species Authentication: Current Prospects and Challenges

Multiplex PCR is a powerful technique that allows simultaneous amplification of multiple DNA targets in a single reaction. It has been widely applied for species authentication in various food matrices, such as meat, fish, dairy, and honey products. Species authentication is important for ensuring food safety, quality, traceability, and compliance with religious, ethical, and environmental standards. However, multiplex PCR also faces some challenges and limitations, such as primer design, optimization, validation, interference, and interpretation of results. This review summarizes the current state of the art of multiplex PCR for species authentication, highlighting its advantages, applications, challenges, and future directions. It also provides some practical guidelines and recommendations for designing and performing multiplex PCR assays for reliable and accurate species identification.

Exploring the role of heterogeneity in quorum sensing cells: a discrete state stochastic approach

Quorum sensing (QS) is a bacterial communication process in which cells detect the local environment and collectively activate gene expression. The transcriptional activator (LuxR) for these genes has a specific allosteric site where the autoinducer (AHL) can bind and turn the activator on. At the single-cell level, the response to gene activation is poorly coordinated among the QS population which leads to heterogeneity in an isogenic population. The paper presents a theoretical framework that uses a discrete-state stochastic approach to address the issue of heterogeneity in the context of DNA target search processes in QS cells and takes into account the relevant physical–chemical processes. This minimal model allows us to understand the molecular mechanisms of the protein target search in the context of cell heterogeneity, and evaluate the analytical expression of the protein search times for the targets on DNA. Specifically, it is shown that the variations in AHL and LuxR binding rates, arising from the asymmetric distribution of LuxR proteins in the cell population, can cause significant variations in the target search on the DNA. It is also found that the intrinsic heterogeneity in the cell population can affect the average search time and this in turn depends on the AHL and LuxR binding rates. Proposed theoretical results have been validated by Monte Carlo simulations.

FLASH Genome Editing Pipeline: An Efficient and High-Throughput Method to Construct Arrayed CRISPR Library for Plant Functional Genomics.

CRISPR/Cas9 genome editing is a revolutionary technology for plant functional genomics and crop breeding. In this system, the Cas9 nuclease is directed by a guide RNA (gRNA) to cut the DNA target and introduce mutation through error-prone DNA break repair. Owing to its simplicity, CRISPR/Cas9-mediated targeted gene knockout is widely used for high-throughput genetic screening in animal cell cultures and bacteria. However, high-throughput genetic screening using CRISPR/Cas9 is still challenging in plants. We recently established a new approach, named the FLASH genome editing pipeline, to construct an arrayed CRISPR library in plants. In this pipeline, a set of 12 PCR fragments with different lengths (referred to as FLASH tags) are used to index the Cas9/gRNA vectors. Subsequently, a mixture of 12 Agrobacterium strains, in which each strain contained a FLASH-tag indexed vector, was transformed into rice plants. As a result, a unique link between the target gene/gRNA and FLASH tag is generated, which allows reading gRNA information in bacterial strains and gene-edited plants using regular PCR and gel electrophoresis. This protocol includes step-by-step instructions for gRNA design, high throughput assembly of FLASH-tag indexed Cas9/gRNA plasmids, Agrobacterium-mediated transformation of 12 indexed plasmids, and fast assignment of target gene information in primary transformants. The arrayed CRISPR library described here is suitable for small- to large-scale genetic screening and allows fast and comprehensive gene function discovery in plants. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Assembly of FLASH-tag-indexed Cas9/gRNA plasmids Basic Protocol 2: Preparation of the Cas9/gRNA plasmid library Basic Protocol 3: Library preparation of Agrobacterium strains and mixing FLASH-tag indexed strains Basic Protocol 4: Grouped transformation and assignments of gRNA information of gene-edited plants.

Targeting DNA Damage Repair and Immune Checkpoint Proteins for Optimizing the Treatment of Endometrial Cancer.

The dependence of cancer cells on the DNA damage response (DDR) pathway for the repair of endogenous- or exogenous-factor-induced DNA damage has been extensively studied in various cancer types, including endometrial cancer (EC). Targeting one or more DNA damage repair protein with small molecules has shown encouraging treatment efficacy in preclinical and clinical models. However, the genes coding for DDR factors are rarely mutated in EC, limiting the utility of DDR inhibitors in this disease. In the current review, we recapitulate the functional role of the DNA repair system in the development and progression of cancer. Importantly, we discuss strategies that target DDR proteins, including PARP, CHK1 and WEE1, as monotherapies or in combination with cytotoxic agents in the treatment of EC and highlight the compounds currently being evaluated for their efficacy in EC in clinic. Recent studies indicate that the application of DNA damage agents in cancer cells leads to the activation of innate and adaptive immune responses; targeting immune checkpoint proteins could overcome the immune suppressive environment in tumors. We further summarize recently revolutionized immunotherapies that have been completed or are now being evaluated for their efficacy in advanced EC and propose future directions for the development of DDR-based cancer therapeutics in the treatment of EC.

Autonomous eDNA collection using an uncrewed surface vessel over a 4200‐km transect of the eastern Pacific Ocean

AbstractThe collection of environmental DNA (eDNA) samples is often laborious, costly, and logistically difficult to accomplish at high frequency in remote locations and over large geographic areas. Here, we addressed those challenges by combining two robotic technologies: an uncrewed surface vessel (USV) fitted with an automated eDNA sample collection device to survey surface waters in the eastern North Pacific Ocean from Alameda, CA to Honolulu, HI. USV Surveyor SD 1200 (Saildrone) carrying the Environmental Sample Processor (ESP) collected 2‐L water samples by filtration followed by RNAlater preservation at regular intervals over a 4200‐km, 29‐day transit. Sixty samples (52 field and 8 controls) were acquired and used to estimate the concentration of specific genes and assess eukaryotic diversity via targeted qPCR and metabarcoding of the cytochrome oxidase subunit I (COI) gene, respectively. Comparisons of control samples revealed important considerations for interpreting results. Samples stored at ambient temperatures onboard Surveyor over the length of the voyage had less total recoverable DNA and specific target gene concentrations compared to the same material immediately flash‐frozen after collection and stored in a laboratory. In contrast, the biodiversity of the COI genes in those samples was similar regardless of sample age and storage condition. COI genes affiliated with 40 eukaryotic phyla were found in native samples collected during the voyage. The distribution and dominance of those phyla varied across different regions, with some taxa spanning large continuous stretches >2000 km, while others were only detected in a single sample. This work highlights the utility and potential of using USVs fitted with autonomous eDNA sample collection devices to improve ocean exploration and support large, basin‐scale, systematic biodiversity surveys. Results of this study also inform future technical considerations for using automated eDNA samplers to acquire material and store it over prolonged periods under prevailing environmental conditions.

Understanding Oligonucleotide Hybridization and the Role of Anchoring on the Single-Walled Carbon Nanotube Corona Phase for Viral Sensing Applications

Semiconducting single-walled carbon nanotubes (SWCNTs) with tailored corona phases (CPs), or surface adsorbed molecules, have emerged as a promising interface for sensing applications. The adsorption of an analyte can be specifically transduced as a modulation of their band gap near-infrared (nIR) photoluminescence (PL). One such CP ideal for this purpose is single-stranded DNA (ssDNA), where subsequent sequence dependent hybridization can result in PL emission wavelength shifts. Due to ssDNA adsorption to the SWCNT surface, the resultant non-canonical hybridization and its effect on SWCNT photophysical properties are not well understood. In this work, we study 20- and 21-mer DNA and RNA hybridization on the complementary ssDNA-SWCNT CP in the context of nucleic acid sensing for SARS-CoV-2 sequences as model analytes. We found that the van’t Hoff transition enthalpy of hybridization on SWCNT CP was -11.9 kJ mol-1, much lower than that of hybridization in solution (-707 kJ mol-1). We used SWCNT solvatochromism to calculate the solvent exposed surface area to indicate successful hybridization. We found that having a 30-mer anchor region in addition to the complementary region significantly improved PL response sensitivity and selectivity, with (GT)15 anchor preferred for RNA targets. Coincubation of ssDNA-SWCNT with analyte at 37°C resulted in faster hybridization kinetics without sacrificing specificity. Other methods aimed to improve CP rearrangement kinetics such as bath sonication and surfactant additions were ineffective. We also determined that target sequence choice is important as secondary structure formation in target is negatively correlated with hybridization. Best performing CPs showed detection limits of 11 nM and 13 nM for DNA and RNA targets respectively. Finally, we simulated sensing conditions using saliva environment, showing sensor compatibility in biofluids. In total, this work elucidates key design features and processing to enable sequence specific hybridization on ssDNA-SWCNT CP.

Development of an Innovative Colorimetric DNA Biosensor Based on Sugar Measurement.

The development of biosensors for target detection plays a crucial role in advancing various fields of bioscience. This work presents the development of a genosensor that exploits the colorimetric phenol-sulfuric acid sugar reaction for the detection of DNA, and RNA as specific targets, and DNA intercalator molecules. The biosensor combines simplicity and reliability to create a novel bioassay for accurate and rapid analysis. A 96-well microplate based on a polystyrene polymer was used as the platform for an unmodified capture DNA immobilization via a silanization process and with (3-Aminopropyl) triethoxysilane (APTES). After that, a hybridization step was carried out to catch the target molecule, followed by adding phenol and sulfuric acid to quantify the amount of DNA or RNA sugar backbone. This reaction generated a yellow-orange color on the wells measured at 490 nm, which was proportional to the target concentration. Under the optimum conditions, a calibration curve was obtained for each target. The developed biosensor demonstrated high sensitivity, good selectivity, and linear response over a wide concentration range for DNA and RNA targets. Additionally, the biosensor was successfully employed for the detection of DNA intercalator agents that inhibited the hybridization of DNA complementary to the immobilized capture DNA. The developed biosensor offers a potential tool for sensitive and selective detection in various applications, including virus diagnosis, genetic analysis, pathogenic bacteria monitoring, and drug discovery.

(Invited) CRISPR-Powered Graphene Electronics for Amplification Free Genotyping and CRISPR Editing Quality Control

The discovery of CRISPR technology has revolutionized the fields of transcriptional activation and repression, genome editing, gene-based therapeutics, and diagnostics. The applications of this technology have been rapidly expanding as researchers continue to discover new Cas enzymes, engineer high fidelity Cas orthologs, and modify and synthesize guide RNAs to efficiently direct these Cas enzymes to their targets. In this talk, we will introduce the first-generation DNA biosensors that combine CRISPR technology with the ultra-sensitivity of graphene-based field effect transistors (gFETs) to detect target DNA sequences within the whole genome without the need for DNA amplification. This technology, termed CRISPR-Chip™, utilizes the genome searching capability of Cas and reprogrammable RNA molecule to unzip the double-stranded DNA and bind to its target. This binding event causes a change in graphene conductivity which can be detected in real-time within the gFET construct. CRISPR-Chip was utilized to detect target genes within clinical samples obtained from patients with Duchenne Muscular Dystrophy (Cover of Nature BME-2019), and single cell point mutations in Sickle cell disease and ALS without the need for amplification (Nature BME 2021), within less than 30 minutes. The applications of this technology platform go beyond diagnostics. CRISPR-Chip can provide greater insights on the mechanism of CRISPR and can lead to safe and more effective utilization of this gene editing technology for therapeutic applications. Figure 1

Characterizing the Non-Covalent Binding of a Pyrene-Derived Linker for DNA Immobilization on Graphene Field-Effect Transistors

Graphene field-effect transistors (G-FETs) constitute an emerging platform for biosensing applications. Most genomic applications with G-FETs use single-stranded DNA (ssDNA) as probes in order to capture a specific target DNA sequence and to detect the corresponding change in the electrical response of the sensor1. Controlling the distribution of DNA probes on the graphene surface is crucial to the sensitivity, selectivity and reproducibility of the sensors1. The most popular immobilization approach uses a non-covalent linker molecule named 1-pyrenebutanoic acid succinimidyl ester (PBASE)1. This bifunctional molecule binds to the graphene surface through non-covalent π-π interactions via its aromatic pyrene group, and on the other end, its succinimidyl ester group can form a covalent bond with amine groups added to the terminal end of ssDNA probes. However, the adsorption process of PBASE molecules is not well controlled, which represents a limitation in the optimization of G-FET biosensors.Here, we present an investigation of the kinetics of the non-covalent adsorption of PBASE on graphene, in order to control the density of ssDNA probes for biosensing applications with G-FETs. We fabricated G-FET sensor arrays using CVD-grown graphene and photolithography techniques, as described previously2. First, we investigated the effect of incubation time on PBASE coverage on graphene, using electrical curves as well as Raman hyperspectral imaging (RIMA). We report a significative and reproducible electrical signature for the PBASE compared to the control without PBASE. We find that this electrical signature appears and saturates quickly compared to the timescales usually reported in the literature. Corroborating results were obtained with RIMA spectroscopy, showing a rapid response of the graphene modes following PBASE incubation. Next, we studied the effect of PBASE accumulation on the assembly of ssDNA probes. We will discuss the combined effect of PBASE accumulation and screening effects in saline buffer on the electrical signature of ssDNA probes. Our results will enable a better control on the non-covalent functionalization of graphene with PBASE for the assembly of various biomolecular probes for biosensing applications.1. Béraud A, Sauvage M, Bazan CM, Tie M, Bencherif A, Bouilly D. Graphene field-effect transistors as bioanalytical sensors: design, operation and performance. Analyst. 2021;(146):403-428.2.Bazan CM, Béraud A, Nguyen M, Bencherif A, Martel R and Bouilly D. Dynamic Gate controlled of Aryldiazonium chemistry on Graphene field-effect transistors. Nano Lett. 2022;22(7):2635-2642.

X-ray Crystallographic Study of Preferred Spacing by the NF-κB p50 Homodimer on κB DNA.

Though originally characterized as an inactive or transcriptionally repressive factor, the NF-κB p50 homodimer has become appreciated as a physiologically relevant driver of specific target gene expression. By virtue of its low affinity for cytoplasmic IκB protein inhibitors, p50 accumulates in the nucleus of resting cells, where it is a binding target for the transcriptional co-activator IκBζ. In this study, we employed X-ray crystallography to analyze the structure of the p50 homodimer on κB DNA from the promoters of human interleukin-6 (IL-6) and neutrophil-gelatinase-associated lipocalin (NGAL) genes, both of which respond to IκBζ. The NF-κB p50 homodimer binds 11-bp on IL-6 κB DNA, while, on NGAL κB DNA, the spacing is 12-bp. This begs the question: what DNA binding mode is preferred by NF-κB p50 homodimer? To address this, we engineered a "Test" κB-like DNA containing the core sequence 5'-GGGGAATTCCCC-3' and determined its X-ray crystal structure in complex with p50. This revealed that, when presented with multiple options, NF-κB p50 homodimer prefers to bind 11-bp, which necessarily imposes asymmetry on the complex despite the symmetry inherent in both the protein and its target DNA, and that the p50 dimerization domain can contact DNA via distinct modes.

BCp12/PLA combination: A novel antibacterial agent targeting Mur family, DNA gyrase and DHFR

The combination of natural antimicrobial peptide BCp12/phenyllatic acid (BCp12/PLA) presents a more efficient antibacterial effect, but its antibacterial mechanism remains unclear. This study studied the synergistic antibacterial mechanism of BCp12 and PLA against S. aureus. The results demonstrated that the BCp12/PLA combination presented a synergistic antibacterial effect against S. aureus, with a fractional inhibitory concentration of 0.05. Furthermore, flow cytometry and scanning electron microscope analysis revealed that BCp12 and PLA synergistically promoted cell membrane disruption compared with the group treated only with one compound, inducing structural cell damage and cytoplasmic leakage. In addition, fluorescence spectroscopy analysis suggested that BCp12 and PLA synergistically influenced genomic DNA. BCp12 and PLA targeted enzymes related to peptidoglycan and DNA synthesis and interacted by hydrogen bonding and hydrophobic interactions with mur enzymes (murC, murD, murE, murF, and murG), dihydrofolate reductase, and DNA gyrase. Additionally, the combined treatment successfully inhibited microbial reproduction in the storage of pasteurized milk, indicating that the combination of BCp12 and PLA can be used as a new preservative strategy in food systems. Overall, this study could provide potential strategies for preventing and controlling foodborne pathogens.

Violet phosphorene nanosheets coupled with CRISPR/Cas12a in a biosensor with a low background signal for onsite detection of tigecycline-resistant hypervirulent Klebsiella pneumoniae

CRISPR/Cas12a-based detection has been widely used for DNA targets, but always suffers from a relatively high background fluorescent signal, which potentially gives false positive results, and limits the accuracy and specificity of detection. To address this problem, violet phosphorene nanosheets (VPNSs) with excellent DNA adsorption and light absorption capacities were synthesized by liquid exfoliation from bulk violet phosphorus. The introduction of VPNSs into CRISPR/Cas12a-based detection decreased the relatively high background fluorescent signal by 32.7 %, and resulted in a background with the relative fluorescence unit of 2590.5 that was no longer visible via a visual readout, demonstrating that VPNSs are efficient fluorescence quenchers. By combining the detection with a smartphone-based fluorescence imaging system (FIS), a biosensor was developed for simultaneous detection of virulence genes iucA, iroB, peg-344, rmpA and rmpA2, and resistance gene tet(A) carried by tigecycline-resistant hypervirulent Klebsiella pneumoniae (HvKP). Upon optimization, the VPNSs-CRISPR/Cas12a-FIS biosensor detected 1 cfu of tigecycline-resistant HvKP in 60 min with a low background signal. The whole process was performed with portable devices including a block heater and an FIS, and the signal could be observed with either a smartphone or the naked eye. The biosensor provides new insights for exploration of low background, multiple target, and onsite analytical methods based on CRISPR/Cas12a.

Morphological and Molecular Identification of Helopeltis Species on Cocoa from Kaliwining Experimental Station, Jember, Indonesia

Severe crop loss of cocoa in Indonesia has long been reported to be caused by Helopeltis. It has been noted that cocoa and Helopeltis have existed in Kaliwining Experimental Station of Indonesian Coffee and Cocoa Research Institute (ICCRI), Jember, Indonesia, for about 100 years, and the species have developed during that time. Our study aimed for examining the Helopeltis species that attacked cocoa in this experimental station both morphologically, morphometrically, and molecularly and to compare the species with the one attacking cocoa in Java and Nusa Tenggara, preserved at the GenBank. Helopeltis was sampled from infested cocoa pods collected representatively from cocoa at the Kaliwining ES and then reared in the Crop Protection Laboratory of ICCRI. Adults of Helopeltis laboratory-reared insects were identified, 30 females 30 males were selected. Weexamined the morphology and morphometry of the samples, and their identifica tion was based on an insect identification key. Morphometry measurement of male and female body parts includes body length, antenna segments, head, eyes, collar, wing, tibia, and femur. In addition, the molecular identification of Helopeltissp. was made through DNA extraction, amplification, and sequence analysis of DNA target regions using the COI part of mitochondrial DNA. External morphological identification, supported by morphometry of the Helopeltis specimen from Kaliwning, the white bands on all femora, body length, and the ratio of the firstantennal segment to the posterior width of the pronotum suggested that the insect belongs to Helopeltis bradyi. Similarly, molecular identification using PCR amplification through specific primers from COI gene sequences confirmed that Helopeltis specimens from Kaliwining are classified as Helopeltis bradyi.

Highly multiplexed targeted sequencing strategy for infectious disease surveillance

BackgroundGlobal efforts to characterize diseases of poverty are hampered by lack of affordable and comprehensive detection platforms, resulting in suboptimal allocation of health care resources and inefficient disease control. Next generation sequencing (NGS) can provide accurate data and high throughput. However, shotgun and metagenome-based NGS approaches are limited by low concentrations of microbial DNA in clinical samples, requirements for tailored sample and library preparations plus extensive bioinformatics analysis. Here, we adapted molecular inversion probes (MIPs) as a cost-effective target enrichment approach to characterize microbial infections from blood samples using short-read sequencing. We designed a probe panel targeting 2 bacterial genera, 21 bacterial and 6 fungi species and 7 antimicrobial resistance markers (AMRs).ResultsOur approach proved to be highly specific to detect down to 1 in a 1000 pathogen DNA targets contained in host DNA. Additionally, we were able to accurately survey pathogens and AMRs in 20 out of 24 samples previously profiled with routine blood culture for sepsis.ConclusionsOverall, our targeted assay identifies microbial pathogens and AMRs with high specificity at high throughput, without the need for extensive sample preparation or bioinformatics analysis, simplifying its application for characterization and surveillance of infectious diseases in medium- to low- resource settings.

Duplex loop-mediated isothermal amplification assay for simultaneous detection of human and human male DNA

ObjectiveScreening of human and human male DNA is necessary for forensic DNA analyses. Although quantitative real-time PCR (qPCR) is commonly used for detecting and quantifying these DNA targets, its use as a screening tool is time-consuming and labor-intensive. To streamline and simplify the screening process, we aimed to develop a duplex loop-mediated isothermal amplification (LAMP) assay capable of simultaneously detecting human and human male DNA in a single tube. We assessed the duplex LAMP assay for forensic application.ResultsFor our duplex LAMP assay, we have utilized two fluorescent probes with HEX and FAM fluorophores to specifically detect human and human male DNA, respectively. The HEX (human target) signal was detected from both the male and female DNA samples, and the FAM (male target) signal was detected from only the male DNA sample. This assay has a sensitivity of 10–1 pg of DNA for both targets. Additionally, we successfully detected the two targets in the DNA samples extracted from forensically relevant body fluids, including blood, saliva, semen, and vaginal secretions.

Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system

CRISPR-Cas adaptive immune systems uptake short 'spacer' sequences from foreign DNA and incorporate them into the host genome to serve as templates for crRNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence-specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn2+ dependent, Cas1 preferentially uses Mg2+ over Mn2+. The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing, making the adaptation module self-reliant for prespacer maturation and directional integration.

Au/NG/SWCNTs/FTO-glass modified electrode based electrochemical biosensor for DNA detection

An easy, quick, and efficient detection of Multidrug Resistance (MDR) of cancer cells at the clinical level is the need of the hour for the effective treatment of leukemia patients. In this study, we reported carbon nanomaterials-based biosensors based on Au nanoparticles (Au-NPs) decorated on Nitrogen-doped Graphene (NG) and Single-Walled Carbon Nanotubes (SWCNTs) for effective, efficient, and sensitive detection of affected DNA. Three Au/NG/SWCNT-based Modified Electrode (ME) were made containing 2, 4, and 6 mg SWCNTs. The thiol group attached at the 5′end was immobilized at ME due to a strong bond between the Au-NPs and thiol group. NG/SWCNTs enhanced the performance of the composite due to its better sensitivity and fast electron transport. Electrochemical Impedance Spectroscopy (EIS), Cyclic Voltammetry (CV), and Differential Potential Voltammetry (DPV) analysis were performed to monitor the performance of the sensor after the hybridization of target DNA onto the capture probe on the modified electrode surface. Different target DNA sequences were used to check the selectivity and sensitivity of the fabricated biosensor. The selectivity performance of prepared DNA sensors was also proved to be good shown by DPV results. The findings of this study provide insights for developing new highly sensitive, selective, and cost-effective portable biosensors for MDR gene detection, which will enable the early diagnosis of cancer more effective at the clinical level. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics dramatically increased DNA attachment quantity and complementary DNA detection sensitivity.