Accurate quantification of DNA using on-site PCR (osPCR) by characterizing DNA amplification at single-molecule resolution.

Despite the need in various applications, accurate quantification of nucleic acids still remains a challenge. The widely-used qPCR has reduced accuracy at ultralow template concentration and is susceptible to nonspecific amplifications. The more recently developed dPCR is costly and cannot handle high-concentration samples. We combine the strengths of qPCR and dPCR by performing PCR in silicon-based microfluidic chips and demonstrate high quantification accuracy in a large concentration range. Importantly, at low template concentration, we observe on-site PCR (osPCR), where only certain sites of the channel show amplification. The sites have almost identical ct values, showing osPCR is a quasi-single molecule phenomenon. Using osPCR, we can measure both the ct values and the absolute concentration of templates in the same reaction. Additionally, osPCR enables identification of each template molecule, allowing removal of nonspecific amplification during quantification and greatly improving quantification accuracy. We develop sectioning algorithm that improves the signal amplitude and demonstrate improved detection of COVID in patient samples.

Fast and efficient template-mediated synthesis of genetic variants.

Efficient methods for the generation of specific mutations enable the study of functional variations in natural populations and lead to advances in genetic engineering applications. Here, we present a new approach, mutagenesis by template-guided amplicon assembly (MEGAA), for the rapid construction of kilobase-sized DNA variants. With this method, many mutations can be generated at a time to a DNA template at more than 90% efficiency per target in a predictable manner. We devised a robust and iterative protocol for an open-source laboratory automation robot that enables desktop production and long-read sequencing validation of variants. Using this system, we demonstrated the construction of 31 natural SARS-CoV2 spike gene variants and 10 recoded Escherichia coli genome fragments, with each 4 kb region containing up to 150 mutations. Furthermore, 125 defined combinatorial adeno-associated virus-2 cap gene variants were easily built using the system, which exhibited viral packaging enhancements of up to 10-fold compared with wild type. Thus, the MEGAA platform enables generation of multi-site sequence variants quickly, cheaply, and in a scalable manner for diverse applications in biotechnology.

A Brief History of the Discovery of Gene Cloning in 1975.

An important but little-known step in the cloning of genes took place in Geneva in 1975. Bypassing the enormous complexity of total genomic DNA, it allowed for the cloning, identification, study, and use of all genes that have been isolated ever since. Bernard Mach was head of the Geneva laboratory where this discovery took place. As interviewed by physician-historian Jacalyn Duffin, he explains the nature of the 1975 procedure and highlights the role that this discovery played in the fields of biology, genetic medicine, and biotechnology.

The genome editing revolution.

A series of spectacular scientific discoveries and technological advances in the second half of the 20th century have provided the basis for the ongoing genome editing revolution. The elucidation of structural and functional features of DNA and RNA was followed by pioneering studies on genome editing: Molecular biotechnology was born. Since then, four decades followed during which progress of scientific insights and technological methods continued at an overwhelming pace. Fundamental insights into microbial host-virus interactions led to the development of tools for genome editing using restriction enzymes or the revolutionary CRISPR-Cas technology. In this review, we provide a historical overview of milestones that led to the genome editing revolution and speculate about future trends in biotechnology.

Impact on touch DNA of an alcohol-based hand sanitizer used in COVID-19 prevention.

In the last years, forensic research has been focused on touch DNA in order to improve its evidential value in criminal activity investigations as well as to understand the variables impacting touch DNA. One of the emerging variables is represented by the use of alcohol-based sanitizers, which was suggested for hand hygiene during the COVID-19 pandemic. The aims of the present study were to assess the effect of a hand sanitizer on touch DNA deposition, transfer, and recovery and also to evaluate STR typing success, quality of DNA profiles, and personal identification. Before and after the use of an alcohol-based hand sanitizer, 20 volunteers deposited on glass surfaces 120 fingerprints, containing skin-derived or salivary DNA. Samples were quantified by real-time quantitative PCR (q-PCR), and 76 samples yielding > 15 pg/µl were typed for 21 autosomal STRs by GlobalFiler® PCR Amplification Kit. DNA profiles were classified into single source, mixed, and inconclusive profiles, and a LR assessment was performed by comparison to the reference samples using LRmix Studio software. After the use of hand sanitizer, samples yielded lower quantities of recovered transferred DNA, especially considering samples containing salivary DNA (p < 0.05 by Friedman test). All the 76 amplified samples (63.3% of the total) showed at least 10 typed loci, and 83-100% of profiles were consistent with the reference ones on the basis of a LR value ≥ 106. Results showed that, although the hand sanitizer reduces the DNA recovering, touch DNA samples might still be useful for forensic personal identification even when hand sanitizers are used.

Simultaneous identification of viruses and viral variants with programmable DNA nanobait.

Respiratory infections are the major cause of death from infectious disease worldwide. Multiplexed diagnostic approaches are essential as many respiratory viruses have indistinguishable symptoms. We created self-assembled DNA nanobait that can simultaneously identify multiple short RNA targets. The nanobait approach relies on specific target selection via toehold-mediated strand displacement and rapid readout via nanopore sensing. Here we show that this platform can concurrently identify several common respiratory viruses, detecting a panel of short targets of viral nucleic acids from multiple viruses. Our nanobait can be easily reprogrammed to discriminate viral variants with single-nucleotide resolution, as we demonstrated for several key SARS-CoV-2 variants. Last, we show that the nanobait discriminates between samples extracted from oropharyngeal swabs from negative- and positive-SARS-CoV-2 patients without preamplification. Our system allows for the multiplexed identification of native RNA molecules, providing a new scalable approach for the diagnostics of multiple respiratory viruses in a single assay.

Bare glassy nanopore for length-resolution reading of PCR amplicons from various pathogenic bacteria and viruses.

In this study, it is confirmed that without addition of organic solvent and embedding polymer hydrogel into glass nanopore, bare glass nanopore can faithfully measure various lengths of DNA duplexes from 200 to 3000 base pairs with 200 base pairs resolution, showing well-separated peak amplitudes of blockage currents. Furthermore, motivated by this readout capability of duplex DNA, amplicons from Polymerase Chain Reaction (PCR) amplification are straightforwardly discriminated by bare glassy nanopore without fluorescent labeling. Except simultaneous discrimination of up to 7 different segments of the same lambda genome, various pathogenic bacteria and viruses including SARS-CoV-2 and its mutants in clinical samples can be discriminated at high resolution. Moreover, quantitative measurement of PCR amplicons is obtained with detection range spanning from 0.75 aM to 7.5 pM and detection limit of 7.5 aM, which reveals that bare glass nanopore can faithfully disclose PCR results without any extra labeling.

Programmable DNA biocomputing circuits for rapid and intelligent screening of SARS-CoV-2 variants.

The frequent emergence of SARS-CoV-2 variants increased viral transmissibility and reduced protection afforded by vaccines. The rapid, multichannel, and intelligent screening of variants is critical to minimizing community transmissions. DNA molecular logic gates have attracted wide attention in recent years due to the powerful information processing capabilities and molecular data biocomputing functions. In this work, some molecular switches (MSs) were connected with each other to implement arbitrary binary functions by emulating the threshold switching of MOS transistors and the decision tree model. Using specific sequences of different SARS-CoV-2 variants as inputs, the MSs net was used to build several molecular biocomputing circuits, including NOT, AND, OR, INHIBIT, XOR, half adder, half subtractor, full adder, and full subtractor. Four fluorophores (FAM, Cy3, ROX, and Cy5) were employed in the logic systems to realize the multichannel monitoring of the logic operation results. The logic response is fast and can be finished with 10 min, which facilitates the rapid wide-population screening for SARS-CoV-2 variants. Importantly, the logic results can be directly observed by the naked eye under a portable UV lamp, thus providing a simple and intelligent method to enable high-frequency point-of-care diagnostics, particularly in low-resource communities.

Switching the Activity of CRISPR/Cas12a Using an Allosteric Inhibitory Aptamer for Biosensing.

The current CRISPR/Cas12a-based diagnostic techniques focus on designing the crRNA or substrate DNA elements to indirectly switch the trans-cleavage activity of Cas12a responsive to target information. Here, we propose the use of an allosteric DNA probe to directly regulate the trans-cleavage activity of Cas12a and present a method for sensing different types of analytes. An allosteric inhibitor probe is rationally designed to couple the target recognition sequence with the inhibitory aptamer of the CRISPR/Cas12a system and enables binding to a specific target to induce the change of conformation, which leads to the loss of its inhibitory function on Cas12a. As a result, the structure-switchable probe can regulate the degree of activity of Cas12a depending on the dose of target. Scalability of our strategy can be achieved by simply replacing the loop domain with different target recognition sequences. The proposed method was validated by detecting adenosine triphosphate and let-7a, giving the detection limits of 490 nM and 26 pM, respectively, and showing an excellent specificity. We believe that this work exploits a viable approach to use the inhibitory aptamer of Cas12a as a regulatory element for biosensing purposes, enriching the arsenal of CRISPR/Cas12a-based methods for molecular diagnostics and spurring further development and application of aptamers of the CRISPR/Cas system.

Development of a PNA-DiSc2 based portable absorbance platform for the detection of pathogen nucleic acids.

We report the development of a portable absorption (PortAbs)-based pathogen nucleic acid detection system using peptide nucleic acid (PNA) and a cyanine dye, DiSc2(5). When the dye binds to the PNA-DNA hybrid, it results in a characteristic ∼110 nm shift in the dye absorbance, which we measure using PortAbs. The protocol involves amplification of the target DNA, PNA-DNA hybridization and dye complexing steps followed by absorption measurement. The system is built using a broad-spectrum photodiode whose output is amplified and then measured by a high resolution (24 or 32 bit) analog-to-digital converter. The excitation pulses of light are delivered by a color-changing LED. The sequence of excitation, measurement and display of results are all controlled by an embedded Raspberry-Pi board (or alternatively a laptop). At higher concentrations of the target amplicon (∼200 ng), the color change can be detected visually. At lower concentrations, PortAbs outperforms a plate reader and can detect target DNA as low as 30 ng or approximately 10 nM which is at least 10 fold better than previously reported studies. We validate the methodology using SARS-CoV-2 clinical samples containing about 1000 copies of the viral RNA and show that the entire workflow takes about 90 min. The cost of the complete standalone system is less than INR 40 000 (approx. 500 USD).

Capturing nucleic acid variants with precision using CRISPR diagnostics.

CRISPR/Cas systems have the ability to precisely target nucleotide sequences and enable their rapid identification and modification. While nucleotide modification has enabled the therapeutic correction of diseases, the process of identifying the target DNA or RNA has greatly expanded the field of molecular diagnostics in recent times. CRISPR-based DNA/RNA detection through programmable nucleic acid binding or cleavage has been demonstrated for a large number of pathogenic and non-pathogenic targets. Combining CRISPR detection with nucleic acid amplification and a terminal signal readout step allowed the development of numerous rapid and robust nucleic acid platforms. Wherever the Cas effector can faithfully distinguish nucleobase variants in the target, the platform can also be extended for sequencing-free rapid variant detection. Some initial PAM disruption-based SNV detection reports were limited to finding or integrating mutated/mismatched nucleotides within the PAM sequences. In this review, we try to summarize the developments made in CRISPR diagnostics (CRISPRDx) to date emphasizing CRISPR-based SNV detection. We also discuss the applications where such diagnostic modalities can be put to use, covering various fields of clinical research, SNV screens, disease genotyping, primary surveillance during microbial infections, agriculture, food safety, and industrial biotechnology. The ease of rapid design and implementation of such multiplexable assays can potentially expand the applications of CRISPRDx in the domain of affinity-based target sequencing, with immense possibilities for low-cost, quick, and widespread usage. In the end, in combination with proximity assays and a suicidal gene approach, CRISPR-based in vivo SNV detection and cancer cell targeting can be formulated as personalized gene therapy.

Nucleic acid amplification with specific signal filtration and magnification for ultrasensitive colorimetric detection.

Recently, sensitive, fast and low cost nucleic acid isothermal amplification technologies (such as loop-mediated isothermal amplification, LAMP) have attracted great attention in the urgent needs of point-of-care testing (POCT) and regular epidemic prevention and control. However, unlike PCR which usually employs TaqMan probe to report specific signals, specific-signal-output strategies in isothermal amplification are immature and visual detection even rare, which limits their popularity in POCT. We hypothesize to address this issue by designing a visual-signal-report system to both filtrate and magnify the target information in isothermal amplification. In this work, we developed a specific signal filtration and magnification colorimetric isothermal sensing platform (SFMC for short) for ultrasensitive detection of DNA and RNA. SFMC consists of two processes: an isothermal amplification with specific signal filtration and a self-replication catalyzed hairpin assembly (SRCHA) for rapid target-specific signal magnification and outputting. With these unique properties, this biosensing platform could detect target DNA as low as 5 copies per reaction and target RNA as low as 10 copies per reaction by naked eyes. Benefited from the excellent colorimetric detection performance, this biosensing platform has been successfully used for African swine fever virus (ASFV) and SARS-CoV-2 detection.

CRISPR/Cas12a system responsive DNA hydrogel for label-free detection of non-glucose targets with a portable personal glucose meter.

In this work, personal glucose meter (PGM) as a portable electrochemical device was utilized for sensitive detection of non-glucose targets: N-gene and PCB77, respectively. DNA hydrogel, which can respond to CRISPR/Cas system, was prepared for label-free encapsulating invertase. In the presence of targets, the repeated sequence for the activation of Cas12a was obtained due to the performance of RCA. Unlike "one-to-one" recognition, activated Cas12a can efficiently cleave multiple single-stranded linker DNAs on DNA hydrogels, thus releasing many invertase that can be used for PGM detection. With the amplification of RCA and CRISPR/Cas system, high detection sensitivity can be obtained even using portable PGM. The detection limits for N-gene and PCB77 were 2.6 fM and 3.2 × 10-5 µg/L, respectively, with high specificity and good practical application performance. The developed biosensor can be used for online monitoring with the merit of low cost, easy operation and can be used for various targets analysis.

Development of an IoT-integrated multiplexed digital PCR system for quantitative detection of infectious diseases.

For rapid detection of the COVID-19 infection, the digital polymerase chain reaction (dPCR) with higher sensitivity and specificity has been presented as a promising method of point-of-care testing (POCT). Unlike the conventional real-time PCR (qPCR), the dPCR system allows absolute quantification of the target DNA without a calibration curve. Although a number of dPCR systems have previously been reported, most of these previous assays lack multiplexing capabilities. As different variants of COVID-19 have rapidly emerged, there is an urgent need for highly specific multiplexed detection systems. Additionally, the advances in the Internet of Things (IoT) technology have enabled the onsite detection of infectious diseases. Here, we present an IoT-integrated multiplexed dPCR (IM-dPCR) system involving sample compartmentalization, DNA amplification, fluorescence imaging, and quantitative analysis. This IM-dPCR system comprises three modules: a plasmonic heating-based thermal cycler, a multi-color fluorescence imaging set-up, and a firmware control module. Combined with a custom-developed smartphone application built on an IoT platform, the IM-dPCR system enabled automatic processing, data collection, and cloud storage. Using a self-priming microfluidic chip, 9 RNA groups (e.g., H1N1, H3N2, IFZ B, DENV2, DENV3, DENV4, OC43, 229E, and NL63) associated with three infectious diseases (e.g., influenza, dengue, and human coronaviruses) were analyzed with higher linearity (>98%) and sensitivity (1 copy per µL). The IM-dPCR system exhibited comparable analytical accuracy to commercial qPCR platforms. Therefore, this IM-dPCR system plays a crucial role in the onsite detection of infectious diseases.

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

A DNA-Cu nanocluster and exonuclease I integrated label-free reporting system for CRISPR/Cas12a-based SARS-CoV-2 detection with minimized background signals.

CRISPR-driven biosensing is developing rapidly, but current studies mostly adopt dye-labeled ssDNA as the signal reporter, which is costly and unstable. Herein, we developed a label-free and low-background reporter for CRISPR/Cas12a signaling by integrating DNA-templated copper nanoclusters (DNA-CuNCs) and exonuclease I (EXO I). The template of the DNA-CuNCs was rationally designed as a ds-/ss-DNA hybrid, ensuring that after a quick and nonpersistent cut of Cas12a, a majority of the template can be digested by EXO I. Based on this novel reporter, a biosensor termed CRISPR-CNS (cost-effective, nimble, and sensitive copper nanocluster sensor integrating CRISPR) was developed. Due to the high signal-to-background ratio of our proposed reporter, CRISPR-CNS shows excellent performances for nucleic acid detection, yielding a detection limit of 20 copies for SARS-CoV-2 RNA. Considering its facile synthesis, robust fluorescence, effective cost, and good sensitivity, this combination shall serve as a highly potential output for CRISPR-based point-of-care testing.

Using deep learning to detect digitally encoded DNA trigger for Trojan malware in Bio-Cyber attacks.

This article uses Deep Learning technologies to safeguard DNA sequencing against Bio-Cyber attacks. We consider a hybrid attack scenario where the payload is encoded into a DNA sequence to activate a Trojan malware implanted in a software tool used in the sequencing pipeline in order to allow the perpetrators to gain control over the resources used in that pipeline during sequence analysis. The scenario considered in the paper is based on perpetrators submitting synthetically engineered DNA samples that contain digitally encoded IP address and port number of the perpetrator's machine in the DNA. Genetic analysis of the sample's DNA will decode the address that is used by the software Trojan malware to activate and trigger a remote connection. This approach can open up to multiple perpetrators to create connections to hijack the DNA sequencing pipeline. As a way of hiding the data, the perpetrators can avoid detection by encoding the address to maximise similarity with genuine DNAs, which we showed previously. However, in this paper we show how Deep Learning can be used to successfully detect and identify the trigger encoded data, in order to protect a DNA sequencing pipeline from Trojan attacks. The result shows nearly up to 100% accuracy in detection in such a novel Trojan attack scenario even after applying fragmentation encryption and steganography on the encoded trigger data. In addition, feasibility of designing and synthesizing encoded DNA for such Trojan payloads is validated by a wet lab experiment.

FRAGLER: A Fragment Recycler Application Enabling Rapid and Scalable Modular DNA Assembly.

Rapid and flexible plasmid construct generation at scale is one of the most limiting first steps in drug discovery projects. These hurdles can partly be overcome by adopting modular DNA design principles, automated sequence fragmentation, and plasmid assembly. To this end we have designed a robust, multimodule golden gate based cloning platform for construct generation with a wide range of applications. The assembly efficiency of the system was validated by splitting sfGFP and sfCherry3C cassettes and expressing them in E. coli followed by fluorometric assessment. To minimize timelines and cost for complex constructs, we developed a software tool named FRAGLER (FRAGment recycLER) that performs codon optimization, multiple sequence alignment, and automated generation of fragments for recycling. To highlight the flexibility and robustness of the platform, we (i) generated plasmids for SarsCoV2 protein reagents, (ii) automated and parallelized assemblies, and (iii) built modular libraries of chimeric antigen receptors (CARs) variants. Applying the new assembly framework, we have greatly streamlined plasmid construction and increased our capacity for rapid generation of complex plasmids.

A cautionary note on altered pace of aging in the COVID-19 era.

Coronavirus disease 2019 (COVID-19) is highly age-dependent due to hi-jacking the molecular control of the immune cells by the severe acute respiratory syndrome-corona virus 2 (SARS-CoV-2) leading to aberrant DNA methylation (DNAm) pattern of blood in comparison to normal individuals. These epigenetic modifications have been linked to perturbations to the epigenetic clock, development of long COVID-19 syndrome, and all-cause mortality risk. I reviewed the effects of COVID-19 on different molecular age markers such as the DNAm, telomere length (TL), and signal joint T-cell receptor excision circle (sjTREC). Integrating the accumulated clinical research data, COVID-19 and novel medical management may alter the pace of aging in adult individuals (<60 years). As such, COVID-19 might be a confounder in epigenetic age estimation similar to life style diversities, pathogens and pathologies which may influence the interpretation of DNAm data. Similarly, the SARS-CoV-2 affects T-lymphocyte function with possible influence on sjTREC levels. In contrast, TL measurements performed years before the SARS-CoV-2 pandemic proved that short TL predisposes to severe COVID- 19 independently from chronological age. However, the persistence of COVID-19 epigenetic scars and the durability of the immune response after vaccination and their effect on the ongoing pace of aging are still unknown. In the light of these data, the heterogeneous nature of the samples in these studies mandates a systematic evaluation of the currrent methods. SARS-CoV-2 may modify the reliability of the age estimation models in real casework because blood is the most common biological sample encountered in forensic contexts.

Telomere G-triplex lights up Thioflavin T for RNA detection: new wine in an old bottle.

Few reports are found working on the features and functions of the human telomere G-triplex (ht-G3) though the telomere G-quadruplex has been intensely studied and widely implemented to develop various biosensors. We herein report that ht-G3 lights up Thioflavin T (ThT) and establish a sensitive biosensing platform for RNA detection by introducing a target recycling strategy. An optimal condition was selected out for ht-G3 to promote ThT to generate a strong fluorescence. Accordingly, an ht-G3-based molecular beacon was successfully designed against the corresponding RNA sequence of the SARS-CoV-2 N-gene. The sensitivity for the non-amplified RNA target achieves 0.01 nM, improved 100 times over the conventional ThT-based method. We believe this ht-G3/ThT-based label-free strategy could be widely applied for RNA detection.