DNA Scissors Research Articles

Gene Editing with Artificial DNA Scissors.

Artificial metallo-nucleases (AMNs) are small molecule DNA cleavage agents, also known as DNA molecular scissors, and represent an important class of chemotherapeutic with high clinical potential. This review provides a primary level of exploration on the concepts key to this area including an introduction to DNA structure, function, recognition, along with damage and repair mechanisms. Building on this foundation, we describe hybrid molecules where AMNs are covalently attached to directing groups that provide molecular scissors with enhanced or sequence specific DNA damaging capabilities. As this research field continues to evolve, understanding the applications of AMNs along with synthetic conjugation strategies can provide the basis for future innovations, particularly for designing new artificial gene editing systems.

Ultrasensitive DNA Origami Plasmon Sensor for Accurate Detection in Circulating Tumor DNAs

AbstractEarly lung cancer screening by computed tomography is hampered by pulmonary nodules caused by massive COVID‐19 infections, necessitating an ultrasensitive approach for the early diagnosis of lung cancers at the single‐base level from circular tumor DNAs (ctDNAs). This study introduces an approach that merges DNA origami and DNA scissors technologies in a framework of surface plasmon resonance (SPR) biosensors. By combining the precision of DNA origami probes with the inherent single‐base resolution of DNA scissors, this method systematically addresses the limitations of conventional SPR techniques, resulting in enhanced detection accuracy. The synergistic interplay between DNA scissors and DNA origami enables the SPR biosensors to achieve unprecedented levels of sensitivity, precision, and practical utility. This efficacy allows the precise identification of mutations, demonstrated here by detection of the T790M mutation in the EGFR gene and the G12C mutation in the KRAS gene of non‐small cell lung cancer patients. With this technique, single‐base resolution as well as near zeptomolar‐level sensitivity is achieved. As a result, this discovery holds significant potential to advance the field of precision diagnostics.

3D walkable DNA gears for ultrasensitive detection of multiple microRNAs in lung cancer cell lysates

As a tumor biomarker with therapeutic application potential, microRNA (miRNA) was crucial for the accurate and sensitive detection of early-stage tumors. Herein, a unique three dimensional (3D) DNA nanomachine (DNM) was created, which was capable detecting lung cancer-related biomarkers miRNA-21, miRNA-205 and miRNA-125b in lung cancer cell lysates with extreme sensitivity. The 3D DNM was composed of DNA scissors and three flexible walkable DNA gears modified with various species of silver nanoclusters (AgNCs). Based on the flexibility of DNA scissors and the walkability of DNA gears, neighboring DNA gears closed the distance between different species of AgNCs by walking in the presence of targets, generating fluorescence resonance energy transfer (FRET) effect and emitting different kinds of fluorescence to complete the highly sensitive detection of single targets and multiple targets. The findings demonstrated that a linear model provided an excellent match for the association between fluorescence signal and target miRNAs. For miRNA-21, miRNA-205, and miRNA-125b, the limits of detection (LODs) (signal/noise = 3) were 4.2 pmol/L (pM), 6.3 pM, and 10.2 pM, respectively. Their recoveries in A549 cell lysate samples ranged from 95.3 to 108.8 % with relative standard deviations of 1.26 %–4.88 %. Satisfactorily, the 3D DNM displayed exceptional analytical performance with high sensitivity and stability, strong specificity and reproducibility, which was triumphantly employed to identify miRNAs in tumor cell lysates, providing a workable technique in creating adaptable nanostructure for dependable bioanalysis and clinical diagnosis of cancer biomarkers.

Identification of RNA Binding Partners of CRISPR-Cas Proteins in Prokaryotes Using RIP-Seq.

CRISPR-Cas systems consist of a complex ribonucleoprotein (RNP) machinery encoded in prokaryotic genomes to confer adaptive immunity against foreign mobile genetic elements. Of these, especially the class 2, Type II CRISPR-Cas9 RNA-guided systems with single protein effector modules have recently received much attention for their application as programmable DNA scissors that can be used for genome editing in eukaryotes. While many studies have concentrated their efforts on improving RNA-mediated DNA targeting with these Type II systems, little is known about the factors that modulate processing or binding of the CRISPR RNA (crRNA) guides and the trans-activating tracrRNA to the nuclease protein Cas9, and whether Cas9 can also potentially interact with other endogenous RNAs encoded within the host genome. Here, we describe RIP-seq as a method to globally identify the direct RNA binding partners of CRISPR-Cas RNPs using the Cas9 nuclease as an example. RIP-seq combines co-immunoprecipitation (coIP) of an epitope-tagged Cas9 followed by isolation and deep sequencing analysis of its co-purified bound RNAs. This method can not only be used to study interactions of Cas9 with its known interaction partners, crRNAs and tracrRNA in native systems, but also to reveal potential additional RNA substrates of Cas9. For example, in RIP-seq analysis of Cas9 from the foodborne pathogen Campylobacter jejuni (CjeCas9), we recently identified several endogenous RNAs bound to CjeCas9 RNP in a crRNA-dependent manner, leading to the discovery of PAM-independent RNA cleavage activity of CjeCas9as well as non-canonical crRNAs. RIP-seq can be easily adapted to any other effector RNP of choice from other CRISPR-Cas systems, allowing for the identification of target RNAs. Deciphering novel RNA-protein interactions for CRISPR-Cas proteins within host bacterial genomes will lead to a better understanding of the molecular mechanisms and functions of these systems and enable us to use the in vivo identified interaction rules as design principles for nucleic acid-targeting applications, fitted to each nuclease of interest.

A Click Chemistry Approach to Developing Molecularly Targeted DNA Scissors

Nucleic acid click chemistry was used to prepare a family of chemically modified triplex forming oligonucleotides (TFOs) for application as a new gene-targeted technology. Azide-bearing phenanthrene ligands-designed to promote triplex stability and copper binding-were 'clicked' to alkyne-modified parallel TFOs. Using this approach, a library of TFO hybrids was prepared and shown to effectively target purine-rich genetic elements in vitro. Several of the hybrids provide significant stabilisation toward melting in parallel triplexes (>20 °C) and DNA damage can be triggered upon copper binding in the presence of added reductant. Therefore, the TFO and 'clicked' ligands work synergistically to provide sequence-selectivity to the copper cutting unit which, in turn, confers high stabilisation to the DNA triplex. To extend the boundaries of this hybrid system further, a click chemistry-based di-copper binding ligand was developed to accommodate designer ancillary ligands such as DPQ and DPPZ. When this ligand was inserted into a TFO, a dramatic improvement in targeted oxidative cleavage is afforded.

APPLICATION OF CRISPR ACTIVATION (CRISPRa) SYSTEM TO ZEBRAFISH (Danio rerio) GENES

CRISPR activation system is part of the function of CRSPR/Cas9 which is used to manipulate certain genes by activating these genes. CRISPR activation utilizes dCas9 (dead-Cas9) or Cas9 which is deactivated as DNA scissors, so that the use of Cas9 for gene activation does not cause targeted DNA chain termination. This research that uses this CRISPR activation is applied to the zebrafish gene (Danio rerio) aims to determine the mRNA level of the targeted gene. In this study, the ASCL1a and BCL6a genes from zebrafish were targeted as the object of study. The results showed that genes from zebrafish that were targeted had an increase in mRNA levels after being activated using CRISPR activation system. Keywords: CRISPR activation system (CRISPRa), zebrafish (Danio rerio), dCas9, ASCL1a gene, BCL6a gene

Reversible and Distance-Controllable DNA Scissor: A Regenerated Electrochemiluminescence Biosensing Platform for Ultrasensitive Detection of MicroRNA.

Reversing the switching of DNA scissors with precisely control remains a compelling goal. Herein, based on strand displacement reaction within single step, the DNA scissor realized reversible switching and further controlled the distance of end strands along the movement of DNA scissor, which has been applied for the development of a regenerated sensing platform for the ultrasensitive detection of microRNA-21 (miRNA-21) with the electrochemiluminescence (ECL) complex (PEI-Ru(II)) as luminophores and diethylenetriamine (DETA) as the coreactant. In the presence of ferrocene-labeled DNA (Fc-DNA), the DETA-labeled DNA scissor clockwise switched to "off" state based on strand displacement reaction, resulting in the significant ECL quenching of Ru(II) system. Next, by using miRNA-21 as the motive fuel, the configuration of DNA scissor could be anticlockwise switched, which significantly enhanced the ECL intensity of Ru(II) complex due to the releasing of Fc-DNA and the proximity between DETA and Ru(II) complex. The reversible switching of DNA scissor led to the remarkably enhancing of ECL signal, realizing ultrasensitive detection of miRNA-21 with an excellent detection limit of 0.17 fM, which was also applied in miRNA detection successfully from different cancer cells. Impressively, the reversible switching of DNA scissor biosensor was able to realize the regeneration of the biosensing platform by adding an additional single stranded DNA (ssDNA) based on strand displacement reaction within a single step, providing a novel concept for constructing simple and sensitive regenerated biosensor.

An exciting time for genome editing: an interview with David R. Liu.

In 1987, several Osaka University researchers discovered a special kind of clustered DNA repeats in bacteria. Within a few years, two other groups independently discovered the same phenomenon but no one knew its function at the time. Only a small handful of scientists studied this property from its discovery in 1987 to 2005. It was then that the function of these DNA repeats, which were named Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), was finally elucidated. Researchers found that CRISPR, when combined with its CRISPR-associated partner (Cas), is crucial for the functioning of the bacterial adaptive immune system against viral phage infection. CRISPR sequences can be transcribed into targeting RNA molecules, and Cas enzymes are guided by these RNAs to cut specific viral DNA loci, rendering resistance against the viral infection.Scientists realized that this natural bacterial immune response system could be engineered to become a powerful genome editing tool. Prior to CRISPR, existing genome editing tools such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) relied solely upon protein–DNA interactions to target an enzyme to specific DNA sequences. The design, engineering and evolution of proteins for various DNA sequences is difficult and time-consuming. In contrast, the CRISPR-Cas system uses Watson–Crick base pairing between a guide RNA and the target DNA to localize the complex to specific DNA sequences. This feature enables users to simply change an RNA sequence to match a DNA target to reposition the whole complex.Since then, numerous talented scientists have headed into this field. Within a single decade, they have developed the CRISPR-Cas system into a powerful genome editing tool and applied it to the editing of microorganisms, plants, animals and even human embryos. David R. Liu, Professor of Harvard University and the Broad Institute, and an investigator of the Howard Hughes Medical Institute, is one of them. One of his major contributions to the field is the development of ‘base editing’. His group engineered the CRISPR system to transform it from being DNA scissors that cut DNA into specific DNA base pair rewriters that directly convert one base pair to a different base pair. This development opens the door to precision genome editing, raising the possibility of treating thousands of genetic diseases that are caused by single point mutations in the human genome. Here, David talks about this exciting time for genome editing.

Synthesis and Evaluation of Artificial DNA Scissors: An Interdisciplinary Undergraduate Experiment

Bridging organic and inorganic chemistry as well as molecular biology, this undergraduate experiment deals with the synthesis of ligands for metal complexes that can cleave DNA. Students gain theoretical and experimental expertise about metal–DNA interactions and the mechanisms behind DNA cleavage. This experiment lays a basis for the understanding and the rational design of artificial DNA scissors and provides insight into interdisciplinary research in the field of bioinorganic chemistry.

Homing endonucleases: DNA scissors on a mission

Buried within the genomes of many microorganisms are genetic elements that encode rare-cutting homing endonucleases that assist in the mobility of the elements that encode them, such as the self-splicing group I and II introns and in some cases inteins. There are several different families of homing endonucleases and their ability to initiate and target specific sequences for lateral transfers makes them attractive reagents for gene targeting. Homing endonucleases have been applied in promoting DNA modification or genome editing such as gene repair or "gene knockouts". This review examines the categories of homing endonucleases that have been described so far and their possible applications to biotechnology. Strategies to engineer homing endonucleases to alter target site specificities will also be addressed. Alternatives to homing endonucleases such as zinc finger nucleases, transcription activator-like effector nucleases, triplex forming oligonucleotide nucleases, and targetrons are also briefly discussed.

Genome modifications in plant cells by custom‐made restriction enzymes

Genome editing, i.e. the ability to mutagenize, insert, delete and replace sequences, in living cells is a powerful and highly desirable method that could potentially revolutionize plant basic research and applied biotechnology. Indeed, various research groups from academia and industry are in a race to devise methods and develop tools that will enable not only site-specific mutagenesis but also controlled foreign DNA integration and replacement of native and transgene sequences by foreign DNA, in living plant cells. In recent years, much of the progress seen in gene targeting in plant cells has been attributed to the development of zinc finger nucleases and other novel restriction enzymes for use as molecular DNA scissors. The induction of double-strand breaks at specific genomic locations by zinc finger nucleases and other novel restriction enzymes results in a wide variety of genetic changes, which range from gene addition to the replacement, deletion and site-specific mutagenesis of endogenous and heterologous genes in living plant cells. In this review, we discuss the principles and tools for restriction enzyme-mediated gene targeting in plant cells, as well as their current and prospective use for gene targeting in model and crop plants.

Targeted genome engineering via zinc finger nucleases

With the development of next-generation sequencing technology, ever-expanding databases of genetic information from various organisms are available to researchers. However, our ability to study the biological meaning of genetic information and to apply our genetic knowledge to produce genetically modified crops and animals is limited, largely due to the lack of molecular tools to manipulate genomes. Recently, targeted cleavage of the genome using engineered DNA scissors called zinc finger nucleases (ZFNs) has successfully supported the precise manipulation of genetic information in various cells, animals, and plants. In this review, we will discuss the development and applications of ZFN technology for genome engineering and highlight recent reports on its use in plants.

Natural and engineered nicking endonucleases--from cleavage mechanism to engineering of strand-specificity

Restriction endonucleases (REases) are highly specific DNA scissors that have facilitated the development of modern molecular biology. Intensive studies of double strand (ds) cleavage activity of Type IIP REases, which recognize 4–8 bp palindromic sequences, have revealed a variety of mechanisms of molecular recognition and catalysis. Less well-studied are REases which cleave only one of the strands of dsDNA, creating a nick instead of a ds break. Naturally occurring nicking endonucleases (NEases) range from frequent cutters such as Nt.CviPII (^CCD; ^ denotes the cleavage site) to rare-cutting homing endonucleases (HEases) such as I-HmuI. In addition to these bona fida NEases, individual subunits of some heterodimeric Type IIS REases have recently been shown to be natural NEases. The discovery and characterization of more REases that recognize asymmetric sequences, particularly Types IIS and IIA REases, has revealed recognition and cleavage mechanisms drastically different from the canonical Type IIP mechanisms, and has allowed researchers to engineer highly strand-specific NEases. Monomeric LAGLIDADG HEases use two separate catalytic sites for cleavage. Exploitation of this characteristic has also resulted in useful nicking HEases. This review aims at providing an overview of the cleavage mechanisms of Types IIS and IIA REases and LAGLIDADG HEases, the engineering of their nicking variants, and the applications of NEases and nicking HEases.

DNA scissors device used to measure MutS binding to DNA mis-pairs.

MutS is a DNA repair protein that recognizes unpaired and bulged bases. When it binds to DNA, it bends the double helix. We have developed a novel DNA-based nanomechanical device that measures the amount of work that a DNA-bending protein can do when it binds to the double helix. The device we report here is a scissors-like device consisting of two double-crossover (DX) molecules connected to each other by a flexible Holliday junction. The two DX components are connected by a double helix that contains the binding site for MutS; when the binding site duplex is bent, the scissors contracts. The two DX molecules are also joined by sticky ends on an edge adjacent to the binding site; the sticky ends can be disrupted if the protein binds with sufficient free energy. Those sticky ends are flanked by a pair of dyes; when the sticky ends are disrupted, the dyes separate, and the fluorescence resonance energy transfer signal can monitor the disruption. The strength of the sticky ends is readily varied, so that the ability of the protein to disrupt them can be quantitated. We use this device to measure work in conjunction with a second device that measures the bending angle resulting from protein binding, so as to calibrate the system. Our data are in good agreement with previous measurements of MutS binding, indicating that this device is able to measure the strength of binding correctly.

Structure and function of nucleases in DNA repair: shape, grip and blade of the DNA scissors.

DNA nucleases catalyze the cleavage of phosphodiester bonds. These enzymes play crucial roles in various DNA repair processes, which involve DNA replication, base excision repair, nucleotide excision repair, mismatch repair, and double strand break repair. In recent years, new nucleases involved in various DNA repair processes have been reported, including the Mus81 : Mms4 (Eme1) complex, which functions during the meiotic phase and the Artemis : DNA-PK complex, which processes a V(D)J recombination intermediate. Defects of these nucleases cause genetic instability or severe immunodeficiency. Thus, structural biology on various nuclease actions is essential for the elucidation of the molecular mechanism of complex DNA repair machinery. Three-dimensional structural information of nucleases is also rapidly accumulating, thus providing important insights into the molecular architectures, as well as the DNA recognition and cleavage mechanisms. This review focuses on the three-dimensional structure-function relationships of nucleases crucial for DNA repair processes.

A Universal, Photocleavable DNA Base: Nitropiperonyl 2‘-Deoxyriboside

A universal, photochemically cleavable DNA base analogue would add desirable versatility to a number of methods in molecular biology. A novel C-nucleoside, nitropiperonyl deoxyriboside (NPdR, P), has been investigated for this purpose. NPdR can be converted to its 5'-DMTr-3'-CE-phosphoramidite and was incorporated into pentacosanucleotides by conventional synthesis techniques. The destabilizing effect on hybrid formation with a complementary strand when this P base opposes A, T, and G was found to be 3-5 kcal/mol, but 9 kcal/mol when it opposes C. Brief irradiation (lambda > 360 nm, 20 min) of DNA containing the P base and piperidine treatment causes strand cleavage giving the 3'- and 5'-phosphates. Two significant recent interests, universal/non-hydrogen-bonding base analogues and photochemical backbone cleavage, have thus been combined in a single molecule that serves as a light-based DNA scissors.