-
Acta Biochimica Et Biophysica Sinica May 2022The rapid development of CRISPR-Cas genome editing tools has greatly changed the way to conduct research and holds tremendous promise for clinical applications. During...
The rapid development of CRISPR-Cas genome editing tools has greatly changed the way to conduct research and holds tremendous promise for clinical applications. During genome editing, CRISPR-Cas enzymes induce DNA breaks at the target sites and subsequently the DNA repair pathways are recruited to generate diverse editing outcomes. Besides off-target cleavage, unwanted editing outcomes including chromosomal structural variations and exogenous DNA integrations have recently raised concerns for clinical safety. To eliminate these unwanted editing byproducts, we need to explore the underlying mechanisms for the formation of diverse editing outcomes from the perspective of DNA repair. Here, we describe the involved DNA repair pathways in sealing Cas enzyme-induced DNA double-stranded breaks and discuss the origins and effects of unwanted editing byproducts on genome stability. Furthermore, we propose the potential risk of inhibiting DNA repair pathways to enhance gene editing. The recent combined studies of DNA repair and CRISPR-Cas editing provide a framework for further optimizing genome editing to enhance editing safety.
Topics: CRISPR-Cas Systems; DNA; DNA Breaks, Double-Stranded; DNA Repair; Gene Editing
PubMed: 35643959
DOI: 10.3724/abbs.2022056 -
Talanta Sep 2022Specific and cost-effective methodologies for human papillomavirus (HPV) gene detection are significant for clinical diagnosis and cancer control. Herein, a label-free...
Specific and cost-effective methodologies for human papillomavirus (HPV) gene detection are significant for clinical diagnosis and cancer control. Herein, a label-free and fluorimetric/colorimetric dual-mode sensing strategy was developed for the quantitative determination of HPV DNA based on the integration of fluorescent DNA-silver nanoclusters (DNA/AgNCs) and G-quadruplex/hemin DNAzyme. The fluorimetric sensing strategy was based on the phenomena that the fluorescence enhancement of DNA/AgNCs obtained in proximity of guanine-rich DNA sequences and the photoinduced electron transfer (PET) effect between the electron donor (DNA/AgNCs) and electron receptor (G-quadruplex/hemin). The colorimetric sensing strategy was relied on the peroxidase-like activity of G-quadruplex/hemin DNAzyme. By integrating DNA/AgNCs and DNAzyme, this dual-mode strategy could produce two independent signals to improve the analytical diversity and accuracy. Under optimized conditions, the fluorimetry and colorimetry of the strategy displayed a linear range of 0.01-4 and 0.02-4 nM, with the low detection limit of 2.3 and 5.2 pM, respectively. Additionally, this dual-mode strategy has been successfully applied to HPV DNA analysis in different real samples with excellent results. Moreover, the sensing platform could be developed for different HPVs DNA assay by only adjusting the recognition sequence, which provided a universal strategy for various kinds of virus analysis.
Topics: Biosensing Techniques; Colorimetry; DNA; DNA, Catalytic; G-Quadruplexes; Hemin; Humans; Nanostructures; Papillomavirus Infections; Silver
PubMed: 35653859
DOI: 10.1016/j.talanta.2022.123554 -
Micron (Oxford, England : 1993) Nov 2022Nanopore-based techniques are widely used owing to their diverse applications such as DNA sequencing, ion detection, gas filtration, protein sequencing, and numerous... (Review)
Review
Nanopore-based techniques are widely used owing to their diverse applications such as DNA sequencing, ion detection, gas filtration, protein sequencing, and numerous other applications. Although commercialized sequencing methods are based on biological nanopores, solid-state nanopore technology is emerging due to its several advantages over biological nanopores, such as its tunable size, chemical and mechanical stability, and possibilities for easy integration with measurement electronics. The unavailability of rapid, low-cost, easy solid-state nanopore fabrication methods with industrial scalability is one of the current bottlenecks in this domain. Among all nanopore fabrication techniques, the Transmission electron microscope (TEM) based fabrication method is frequently used in research labs due to its capability of drilling and tuning nanopores with high accuracy. Given that there are no other methods capable of imaging and fabricating nanopores simultaneously, it is important to discuss the related methods and protocols of TEM. This review focuses on the various aspects of nanopore technology using TEM, from pore fabrication to imaging. Hybrid nanopores are also emerging, which combine the benefits of biological and solid-state nanopores. These can be formed by integrating DNA origami with solid-state nanopores. Creating and imaging DNA origami structures also presents several challenges. We also review DNA origami imaging using conventional TEM. We hope that this review will provide a one-stop reference to TEM applications on solid-state nanopores from fabrication to bioimaging and boost further research in this area.
Topics: DNA; Nanopores; Nanotechnology; Sequence Analysis, DNA
PubMed: 36081256
DOI: 10.1016/j.micron.2022.103347 -
Gene Therapy Jun 2022While generally referred to as "non-integrating" vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous)...
While generally referred to as "non-integrating" vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous) recombination. The present study provides a quantitative assessment of the potential integration frequency of adenovirus 5 (Ad5)-based vectors following intravenous injection in mice, a common route of administration in gene therapy applications particularly for transgene expression in liver. We examined the uptake level and persistence in liver of first generation (FG) and helper-dependent (HD) Ad5 vectors containing the mouse leptin transgene. As expected, the persistence of the HD vector was markedly higher than that of the FG vector. For both vectors, the majority of the vector DNA remained extrachromosomal and predominantly in the form of episomal monomers. However, using a quantitative gel-purification-based integration assay, a portion of the detectable vector was found to be associated with high molecular weight (HMW) genomic DNA, indicating potential integration with a frequency of up to ~44 and 7000 integration events per μg cellular genomic DNA (or ~0.0003 and 0.05 integrations per cell, respectively) for the FG and HD Ad5 vectors, respectively, following intravenous injection of 1 × 10 virus particles. To confirm integration occurred (versus residual episomal vector DNA co-purifying with genomic DNA), we characterized nine independent integration events using Repeat-Anchored Integration Capture (RAIC) PCR. Sequencing of the insertion sites suggests that both of the vectors integrate randomly, but within short segments of homology between the vector breakpoint and the insertion site. Eight of the nine integrations were in intergenic DNA and one was within an intron. These findings represent the first quantitative assessment and characterization of Ad5 vector integration following intravenous administration in vivo in wild-type mice.
Topics: Adenoviridae; Animals; DNA; Genetic Vectors; Genomics; Injections, Intravenous; Liver; Mice
PubMed: 34404916
DOI: 10.1038/s41434-021-00278-2 -
Nature Plants Oct 2016Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then...
Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown. Here we demonstrate that in Arabidopsis thaliana T-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQ mutant plants (which have mutated Pol θ), although susceptible to Agrobacterium infection, are resistant to T-DNA integration. Characterization of >10,000 T-DNA-plant genome junctions reveals a distinct signature of Pol θ action and also indicates that 3' end capture at genomic breaks is the prevalent mechanism of T-DNA integration. The primer-template switching ability of Pol θ can explain the molecular patchwork known as filler DNA that is frequently observed at sites of integration. T-DNA integration signatures in other plant species closely resemble those of Arabidopsis, suggesting that Pol-θ-mediated integration is evolutionarily conserved. Thus, Pol θ provides the mechanism for T-DNA random integration into the plant genome, demonstrating a potential to disrupt random integration so as to improve the quality and biosafety of plant transgenesis.
Topics: Arabidopsis; Arabidopsis Proteins; DNA Repair; DNA, Bacterial; DNA, Plant; DNA-Directed DNA Polymerase; Mutation; DNA Polymerase theta
PubMed: 27797358
DOI: 10.1038/nplants.2016.164 -
Molecules (Basel, Switzerland) Mar 2021The predictable nature of deoxyribonucleic acid (DNA) interactions enables assembly of DNA into almost any arbitrary shape with programmable features of nanometer... (Review)
Review
The predictable nature of deoxyribonucleic acid (DNA) interactions enables assembly of DNA into almost any arbitrary shape with programmable features of nanometer precision. The recent progress of DNA nanotechnology has allowed production of an even wider gamut of possible shapes with high-yield and error-free assembly processes. Most of these structures are, however, limited in size to a nanometer scale. To overcome this limitation, a plethora of studies has been carried out to form larger structures using DNA assemblies as building blocks or tiles. Therefore, DNA tiles have become one of the most widely used building blocks for engineering large, intricate structures with nanometer precision. To create even larger assemblies with highly organized patterns, scientists have developed a variety of structural design principles and assembly methods. This review first summarizes currently available DNA tile toolboxes and the basic principles of lattice formation and hierarchical self-assembly using DNA tiles. Special emphasis is given to the forces involved in the assembly process in liquid-liquid and at solid-liquid interfaces, and how to master them to reach the optimum balance between the involved interactions for successful self-assembly. In addition, we focus on the recent approaches that have shown great potential for the controlled immobilization and positioning of DNA nanostructures on different surfaces. The ability to position DNA objects in a controllable manner on technologically relevant surfaces is one step forward towards the integration of DNA-based materials into nanoelectronic and sensor devices.
Topics: DNA; Nanostructures; Nanotechnology
PubMed: 33801952
DOI: 10.3390/molecules26061502 -
Molecular Cell May 2022Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with... (Review)
Review
Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with biophysical tools allows sequence-dependent mechanistic studies. Using the millions of DNA clusters that are generated during sequencing to perform high-throughput binding affinity and kinetics measurements enabled the construction of energy landscapes in sequence space, uncovering relationships between sequence, structure, and function. Here, we review the approaches to perform ensemble fluorescence experiments on next-generation sequencing chips for variations of DNA, RNA, and protein sequences. As the next step, we anticipate that these fluorescence experiments will be pushed to the single-molecule level, which can directly uncover kinetics and molecular heterogeneity in an unprecedented high-throughput fashion. Molecular biophysics in sequence space, both at the ensemble and single-molecule level, leads to new mechanistic insights. The wide spectrum of applications in biology and medicine ranges from the fundamental understanding of evolutionary pathways to the development of new therapeutics.
Topics: Biophysics; DNA; High-Throughput Nucleotide Sequencing; Molecular Biology; Sequence Analysis, DNA
PubMed: 35561688
DOI: 10.1016/j.molcel.2022.04.024 -
ACS Nano Feb 2020Cells often spatially organize biomolecules to regulate biological interactions. Synthetic mimicry of complex spatial organization may provide a route to similar levels...
Cells often spatially organize biomolecules to regulate biological interactions. Synthetic mimicry of complex spatial organization may provide a route to similar levels of control for artificial systems. As a proof-of-principle, we constructed an RNA-extruding nanofactory using a DNA-origami barrel with an outer diameter of 60 nm as a chassis for integrated rolling-circle transcription and processing of RNA through spatial organization of DNA templates, RNA polymerases, and RNA endonucleases. The incorporation efficiency of molecular components was quantified to be roughly 50% on designed sites within the DNA-origami chassis. Each integrated nanofactory with RNA-producing units, composed of DNA templates and RNA polymerases, produced 100 copies of target RNA in 30 min on average. Further integration of RNA endonucleases that cleave rolling-circle transcripts from concatemers into monomers resulted in 30% processing efficiency. Disabling spatial organization of molecular components on DNA origami resulted in suppression of RNA production as well as processing.
Topics: DNA; DNA-Directed RNA Polymerases; Endoribonucleases; Nanotechnology; Particle Size; RNA; Surface Properties
PubMed: 31922721
DOI: 10.1021/acsnano.9b06466 -
Methods in Molecular Biology (Clifton,... 2022PTO-QuickStep is a quick and easy molecular cloning technique that allows seamless point integration of a DNA fragment, encoding either a tag or a protein, into any...
PTO-QuickStep is a quick and easy molecular cloning technique that allows seamless point integration of a DNA fragment, encoding either a tag or a protein, into any position within a target plasmid. The entire process is conducted in a time-efficient and cost-effective manner, without the need of DNA gel purification and enzymatic restriction and ligation. PTO-QuickStep further innovates protein engineering by providing the possibility of integrating a random mutagenesis step (e.g., error-prone PCR) into the workflow, without compromising the time duration required. Random mutagenesis libraries can be quickly and efficiently cloned into a plasmid of interest, thereby accelerating directed evolution. On top of that, PTO-QuickStep can be utilized for rapid integration of noncoding DNA fragments to modify existing plasmids, making it an excellent tool for synthetic biologists.
Topics: Cloning, Molecular; DNA; Gene Library; Mutagenesis; Plasmids; Polymerase Chain Reaction
PubMed: 35727447
DOI: 10.1007/978-1-0716-2152-3_8 -
FEMS Microbiology Reviews Jul 2017Integrative and conjugative elements (ICEs) are widespread mobile DNA that transmit both vertically, in a host-integrated state, and horizontally, through excision and... (Review)
Review
Integrative and conjugative elements (ICEs) are widespread mobile DNA that transmit both vertically, in a host-integrated state, and horizontally, through excision and transfer to new recipients. Different families of ICEs have been discovered with more or less restricted host ranges, which operate by similar mechanisms but differ in regulatory networks, evolutionary origin and the types of variable genes they contribute to the host. Based on reviewing recent experimental data, we propose a general model of ICE life style that explains the transition between vertical and horizontal transmission as a result of a bistable decision in the ICE-host partnership. In the large majority of cells, the ICE remains silent and integrated, but hidden at low to very low frequencies in the population specialized host cells appear in which the ICE starts its process of horizontal transmission. This bistable process leads to host cell differentiation, ICE excision and transfer, when suitable recipients are present. The ratio of ICE bistability (i.e. ratio of horizontal to vertical transmission) is the outcome of a balance between fitness costs imposed by the ICE horizontal transmission process on the host cell, and selection for ICE distribution (i.e. ICE 'fitness'). From this emerges a picture of ICEs as elements that have adapted to a mostly confined life style within their host, but with a very effective and dynamic transfer from a subpopulation of dedicated cells.
Topics: DNA Transposable Elements; Host-Pathogen Interactions
PubMed: 28369623
DOI: 10.1093/femsre/fux008