Site-specific Cleavage Research Articles

Repair of a Site-Specific DNA Cleavage: Old-School Lessons for Cas9-Mediated Gene Editing.

CRISPR/Cas9-mediated gene editing may involve nonhomologous end-joining to create various insertion/deletions (indels) or may employ homologous recombination to modify precisely the target DNA sequence. Our understanding of these processes has been guided by earlier studies using other site-specific endonucleases, both in model organisms such as budding yeast and in mammalian cells. We briefly review what has been gleaned from such studies using the HO and I-SceI endonucleases and how these findings guide current gene editing strategies.

Endonucleolytic cleavage in the expansion segment 7 of 25S rRNA is an early marker of low-level oxidative stress in yeast

The ability to detect and respond to oxidative stress is crucial to the survival of living organisms. In cells, sensing of increased levels of reactive oxygen species (ROS) activates many defensive mechanisms that limit or repair damage to cell components. The ROS-signaling responses necessary for cell survival under oxidative stress conditions remain incompletely understood, especially for the translational machinery. Here, we found that drug treatments or a genetic deficiency in the thioredoxin system that increase levels of endogenous hydrogen peroxide in the yeast Saccharomyces cerevisiae promote site-specific endonucleolytic cleavage in 25S ribosomal RNA (rRNA) adjacent to the c loop of the expansion segment 7 (ES7), a putative regulatory region located on the surface of the 60S ribosomal subunit. Our data also show that ES7c is cleaved at early stages of the gene expression program that enables cells to successfully counteract oxidative stress and is not a prerequisite or consequence of apoptosis. Moreover, the 60S subunits containing ES7c-cleaved rRNA cofractionate with intact subunits in sucrose gradients and repopulate polysomes after a short starvation-induced translational block, indicating their active role in translation. These results demonstrate that ES7c cleavage in rRNA is an early and sensitive marker of increased ROS levels in yeast cells and suggest that changes in ribosomes may be involved in the adaptive response to oxidative stress.

Pushing the haystack aside for efficient gene targeting in human cells.

Gene targeting, i.e., the disruption of a specific gene via homologous recombination, is one of the key techniques to study gene function. However, researchers have faced the difficulty that, in human cells, the frequency of correct targeted integration is quite low compared to non-targeted (or random) integration. Adachi and colleagues investigated the factors that influence the ratio of targeted integration to non-targeted integration.

Rapid RNase L–driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery

Mammalian cells respond to double-stranded RNA (dsRNA) by activating a translation-inhibiting endoribonuclease, RNase L. Consensus in the field indicates that RNase L arrests protein synthesis by degrading ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs). However, here we provide evidence for a different and far more efficient mechanism. By sequencing abundant RNA fragments generated by RNase L in human cells, we identify site-specific cleavage of two groups of noncoding RNAs: Y-RNAs, whose function is poorly understood, and cytosolic tRNAs, which are essential for translation. Quantitative analysis of human RNA cleavage versus nascent protein synthesis in lung carcinoma cells shows that RNase L stops global translation when tRNAs, as well as rRNAs and mRNAs, are still intact. Therefore, RNase L does not have to degrade the translation machinery to stop protein synthesis. Our data point to a rapid mechanism that transforms a subtle RNA cleavage into a cell-wide translation arrest.

Interaction of Zn(II)bleomycin-A2 and Zn(II)peplomycin with a DNA hairpin containing the 5'-GT-3' binding site in comparison with the 5'-GC-3' binding site studied by NMR spectroscopy.

Bleomycins are a group of glycopeptide antibiotics synthesized by Streptomyces verticillus that are widely used for the treatment of various neoplastic diseases. These antibiotics have the ability to chelate a metal center, mainly Fe(II), and cause site-specific DNA cleavage. Bleomycins are differentiated by their C-terminal regions. Although this antibiotic family is a successful course of treatment for some types of cancers, it is known to cause pulmonary fibrosis. Previous studies have identified that bleomycin-related pulmonary toxicity is linked to the C-terminal region of these drugs. This region has been shown to closely interact with DNA. We examined the binding of Zn(II)peplomycin and Zn(II)bleomycin-A2 to a DNA hairpin of sequence 5'-CCAGTATTTTTACTGG-3', containing the binding site 5'-GT-3', and compared the results with those obtained from our studies of the same MBLMs bound to a DNA hairpin containing the binding site 5'-GC-3'. We provide evidence that the DNA base sequence has a strong impact in the final structure of the drug-target complex.

A positive readout single transcript reporter for site-specific mRNA cleavage.

Cleavage of mRNA molecules causes their rapid degradation, thereby playing an important role in regulation of gene expression and host genome defense from viruses and transposons in bacterial and eukaryotic cells. Current negative-readout, and repressor-based positive-readout reporters of mRNA degradation have limitations. Here we report the development of a single transcript that acts as a positive reporter of mRNA cleavage. We show that placement of bacterial CopT and CopA hairpins into the 5′ UTR and 3′ UTR of an mRNA results in inhibition of translation of the intervening coding sequence in Drosophila. An internal poly(A) tract inserted downstream of the coding sequence stabilizes transcripts cut within the 3′ UTR. When these components are combined in a transcript in which targets sites for RNA cleavage are placed between the poly(A) tract and CopA, cleavage results in translational activation, providing a single transcript-based method of sensing mRNA cleavage with a positive readout.

Convective-heating thermal decomposition/digestion of peptides and proteins on surfaces

A site specific cleavage at the N-terminus of the amino acid cysteine (Cys, C) was induced on peptides and proteins deposited on surfaces via convective heating (hot air) at temperatures between 220 and 270°C for 30s. Protein and peptide samples studied were prepared as thin films in order to simulate biological tissue sections. Specifically, a stream of hot air was applied to biological samples on surfaces. The site-specific bond cleavage at Cys was found to be initiated by a loss of a hydrogen sulfide molecule from its side chain, followed by the formation of an intermediate dehydroalanine, rearrangement and water addition. This thermal decomposition process induces a cleavage of the bond between the nitrogen and α-carbon (N-terminus) of Cys. The N-terminal product, with a mass change of −1Da, shows an amidated C-terminus, while the C-terminal product has a net −33Da mass change. Thermal decomposition products of peptides and proteins were analyzed by direct insertion probe electron ionization (DIP-EI)-mass spectrometry (MS), electrospray ionization (ESI)-MS and tandem MS (MS/MS), matrix-assisted laser desorption/ionization (MALDI)-MS, and MALDI-MS/MS analyses. Molecular water was found to have little influence on the elimination of hydrogen sulfide from the intact peptide, but essential in the final cleavage step. This thermal decomposition and digestion (TDD) reaction was observed in peptide and protein samples ranging in molecular weights from 770Da to 66kDa. The cleavage specificity and speed (30s) of the convective-heating TDD method make it a viable approach for the rapid analysis of proteins in tissue or biofilm samples as part of a bottom-up in situ (or on-tissue) protein identification protocol by MALDI-MS/MS.

Structural Basis for Substrate Helix Remodeling and Cleavage Loop Activation in the Varkud Satellite Ribozyme.

The Varkud satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the ribozyme, resulting in a rearrangement of the substrate helix register into a so-called "shifted" conformation that is critical for substrate binding and activation. We report a 3.3 Å crystal structure of the complete ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and ribozyme cleavage.

Expression of Flp Protein in a Baculovirus/Insect Cell System for Biotechnological Applications.

The Saccharomyces cerevisiae Flp protein is a site-specific recombinase that recognizes and binds to the Flp recognition target (FRT) site, a specific sequence comprised of at least two inverted repeats separated by a spacer. Binding of four monomers of Flp is required to mediate recombination between two FRT sites. Because of its site-specific cleavage characteristics, Flp has been established as a genome engineering tool. Amongst others, Flp is used to direct insertion of genes of interest into eukaryotic cells based on single and double FRT sites. A Flp-encoding plasmid is thereby typically cotransfected with an FRT-harboring donor plasmid. Moreover, Flp can be used to excise DNA sequences that are flanked by FRT sites. Therefore, the aim of this study was to determine whether Flp protein and its step-arrest mutant, FlpH305L, recombinantly expressed in insect cells, can be used for biotechnological applications. Using a baculovirus system, the proteins were expressed as C-terminally 3 × FLAG-tagged proteins and were purified by anti-FLAG affinity selection. As demonstrated by electrophoretic mobility shift assays (EMSAs), purified Flp and FlpH305L bind to FRT-containing DNA. Furthermore, using a cell assay, purified Flp was shown to be active in recombination and to mediate efficient insertion of a donor plasmid into the genome of target cells. Thus, these proteins can be used for applications such as DNA-binding assays, in vitro recombination, or genome engineering.

Splicing stimulates siRNA formation at Drosophila DNA double-strand breaks.

DNA double-strand breaks trigger the production of locus-derived siRNAs in fruit flies, human cells and plants. At least in flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DNA ends can generate matching sense and antisense transcripts. We performed a genome-wide RNAi-screen in cultured Drosophila cells, which revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation. We validated this observation through site-specific DNA cleavage with CRISPR-cas9 followed by deep sequencing of small RNAs. DNA breaks in intron-less genes or upstream of a gene’s first intron did not efficiently trigger siRNA production. When DNA double-strand breaks were induced downstream of an intron, however, this led to robust siRNA generation. Furthermore, a downstream break slowed down splicing of the upstream intron and a detailed analysis of siRNA coverage at the targeted locus revealed that unspliced pre-mRNA contributes the sense strand to the siRNA precursor. Since splicing factors are stimulating the response but unspliced transcripts are entering the siRNA biogenesis, the spliceosome is apparently stalled in a pre-catalytic state and serves as a signaling hub. We conclude that convergent transcription at DNA breaks is stimulated by a splicing dependent control process. The resulting double-stranded RNA is converted into siRNAs that instruct the degradation of cognate mRNAs. In addition to a potential role in DNA repair, the break-induced transcription may thus be a means to cull improper RNAs from the transcriptome of Drosophila melanogaster. Since the splicing factors identified in our screen also stimulated siRNA production from high copy transgenes, it is possible that this surveillance mechanism serves in genome defense beyond DNA double-strand breaks.

Comparison of DNA fragments as donor DNAs upon sequence conversion of cleaved target DNA

ABSTRACTPinpoint sequence alteration (genome editing) by the combination of the site-specific cleavage of a target DNA and a donor nucleic acid has attracted much attention and the sequence of the target DNA is expected to be changed to that of a donor nucleic acid. In most cases, oligodeoxyribonucleotides (ODNs) and plasmid DNAs have been used as donors. However, a several hundred-base single-stranded (ss) DNA fragment and a 5′-tailed duplex (TD) accomplished the desired sequence changes without DNA cleavage, and might serve as better donors for the cleaved target DNA than ODNs and plasmid DNAs. In this study, sequence conversion efficiencies were compared with various donor DNAs in model sequence alteration experiments, using episomal DNA. The efficiencies with the ss and TD fragments were higher than those with the ODN and plasmid DNA. The sequence change by the TD seemed somewhat less efficient but slightly more accurate than that by the ss DNA fragment. These results suggested that the ss and TD fragments are better donors for targeted sequence alteration.

Structural and Biochemical Properties of Novel Self-Cleaving Ribozymes.

Fourteen well-defined ribozyme classes have been identified to date, among which nine are site-specific self-cleaving ribozymes. Very recently, small self-cleaving ribozymes have attracted renewed interest in their structure, biochemistry, and biological function since the discovery, during the last three years, of four novel ribozymes, termed twister, twister sister, pistol, and hatchet. In this review, we mainly address the structure, biochemistry, and catalytic mechanism of the novel ribozymes. They are characterized by distinct active site architectures and divergent, but similar, biochemical properties. The cleavage activities of the ribozymes are highly dependent upon divalent cations, pH, and base-specific mutations, which can cause changes in the nucleotide arrangement and/or electrostatic potential around the cleavage site. It is most likely that a guanine and adenine in close proximity of the cleavage site are involved in general acid-base catalysis. In addition, metal ions appear to play a structural rather than catalytic role although some of their crystal structures have shown a direct metal ion coordination to a non-bridging phosphate oxygen at the cleavage site. Collectively, the structural and biochemical data of the four newest ribozymes could contribute to advance our mechanistic understanding of how self-cleaving ribozymes accomplish their efficient site-specific RNA cleavages.

Quantum dots-based fluorescence resonance energy transfer biosensor for monitoring cell apoptosis.

The development of advanced methods for accurately monitoring cell apoptosis has extensive significance in the diagnostic and pharmaceutical fields. In this study, we developed a rapid, sensitive and selective approach for the detection of cell apoptosis by combining the site-specific recognition and cleavage of the DEVD-peptide with quantum dots (QDs)-based fluorescence resonance energy transfer (FRET). Firstly, biotin-peptide was conjugated on the surface of AuNPs to form AuNPs-pep through the formation of an Au-S bond. Then, AuNPs-pep-QDs nanoprobe was obtained through the connection between AuNPs-pep and QDs. FRET is on and the fluorescence of QDs is quenched at this point. The evidence of UV-vis spectra, transmission electron microscopy (TEM), and Fourier transform infrared (FT-IR) spectroscopy revealed that the connection was successful. Upon the addition of apoptosis cell lysis solution, peptide was cleaved by caspase-3, and AuNPs was dissociated from the QDs. At this time, FRET is off, and thus the QDs fluorescence was recovered. The experimental conditions were optimized in terms of ratio of peptide to AuNPs, buffer solution, and the temperature of conjugation and enzyme reaction. The biosensor was successfully applied to distinguishing apoptosis cells and normal cells within 2h. This study demonstrated that the biosensor could be utilized to evaluate anticancer drugs.

Intra-endosomal trafficking mediated by lysobisphosphatidic acid contributes to intracellular release of phosphorothioate-modified antisense oligonucleotides

Antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages are broadly used as research tools and therapeutic agents. Chemically modified PS-ASOs can mediate efficient target reduction by site-specific cleavage of RNA through RNase H1. PS-ASOs are known to be internalized via a number of endocytotic pathways and are released from membrane-enclosed endocytotic organelles, mainly late endosomes (LEs). This study was focused on the details of PS-ASO trafficking through endocytic pathways. It was found that lysobisphosphatidic acid (LBPA) is required for release of PS-ASOs from LEs. PS-ASOs exited early endosomes (EEs) rapidly after internalization and became co-localized with LBPA by 2 hours in LEs. Inside LEs, PS-ASOs and LBPA were co-localized in punctate, dot-like structures, likely intraluminal vesicles (ILVs). Deactivation of LBPA using anti-LBPA antibody significantly decreased PS-ASO activities without affecting total PS-ASO uptake. Reduction of Alix also substantially decreased PS-ASO activities without affecting total PS-ASO uptake. Furthermore, Alix reduction decreased LBPA levels and limited co-localization of LBPA with PS-ASOs at ILVs inside LEs. Thus, the fusion properties of ILVs, which are supported by LBPA, contribute to PS-ASO intracellular release from LEs.

A stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair

The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) after aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway or, alternatively, for enhancing homology-directed repair to facilitate the generation of a specific mutation (or "knock-in"). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA activity. Reliable methods for enriching and identifying desired mutants are also lacking. Here we describe a method using the Piggybac transposon for stable genomic integration of an H2B-GFP reporter or a hygromycin resistance gene for assaying Cas9 target cleavage and homology-directed repair. The H2B-GFP fusion protein provides increased stability and an obvious pattern of nuclear localization. This method, called SRIRACCHA (i.e. a stable, but reversible, integrated reporter for assaying CRISPR/Cas-stimulated HDR activity), enables the enrichment of mutants via selection of GFP-positive or hygromycin-resistant mammalian cells (immortalized or non-immortalized) as a surrogate for the modification of the endogenous target site. Currently available hyperactive Piggybac transposase mutants allow both delivery and removal of the surrogate reporters, with minimal risk of generating undesirable mutations. This assay permits rapid screening for efficient guide RNAs and the accelerated identification of mutant clones and is applicable to many cell types. We foresee the utility of this approach in contexts in which the maintenance of genomic integrity is essential, for example, when engineering cells for therapeutic purposes.

Chiral photochemical scissors: Toward site specific cleavage of proteins with light

Abstract A powerful strategy to photocleave the protein backbone at a single site, developed over nearly two decades, is described here. The remarkable ability to target one or even a couple of sites on a large protein with a small molecule, under photochemical control, is a considerable challenge. Systematic modification of the structure of the photoreagent provides valuable information on the binding site recognition as well as the mechanism of the photocleavage reaction. Some factors that impact the photocleavage reaction include the exact location of the probe binding site on the protein, conformations of the bound probe, protein size, functional groups present on the probe that interact with the protein surroundings either in a favorable or unfavorable manner, overall charge on the photoreagent, and photophysical/photochemical properties of the probe. The protein photocleavage studies were preceded by detailed binding studies by a variety of spectroscopic methods. Methods as simple as absorption and fluorescence spectroscopies or more sophisticated circular dichroism spectroscopy were used. Conclusions that are most consistent with the binding data indicated a single binding site on most proteins, irrespective of the probe or the protein, with only one exception noted so far. Such high selectivity for targeting a binding site on a biomolecule by a small molecule is remarkable. Photocleavage studies and laser flash photolysis studies were used to probe the photochemical peptide bond cleavage reactions, which indicated the important role of aromatic cation radicals in the mechanistic path. Our work focused mainly on few generic proteins serum albumin, lysozyme, and avidin; but may be extended to other proteins with appropriate modifications. This article strives to provide some guidance on such design strategies for other proteins or other photoreagents. Molecular modeling studies clearly supported our conclusions and the methods described here could be useful in a rational design of novel photoreagents to target desired sites on proteins in future studies.

The Structural and Mechanistic Origins of Catalysis in Nucleolytic Ribozymes

The nucleolytic ribozymes are a structurally diverse and widespread group of catalytic RNA species. They accelerate transesterification reactions around a million fold, resulting in the site-specific cleavage or ligation of RNA. The potential entities that can participate are the nucleobases, 2′-hydroxyl groups and hydrated metal ions. Probable catalytic strategies are the facilitation of in-line attack, stabilization of the phosphorane transition state, deprotonation of the nucleophile and protonation of the oxyanion leaving group (these last two being general base-acid catalysis).While much of the above was studied in ribozymes such as hammerhead, hairpin and VS, these principles are well illustrated by newer ribozymes. We have solved the crystal structure of the twister ribozyme, which adopts a double pseudoknot fold with a central active site. This well illustrates the four strategies summarized above. We have recently solved the structure of a new ribozyme, that appears mechanistically very different, with a key role for a bound metal ion where an inner-sphere water molecule acts as a general base.

Exploring Mechanisms of Site Specific DNA Cleavage with Single Molecule Sensitivity

Exploring Mechanisms of Site Specific DNA Cleavage with Single Molecule Sensitivity

Genome Editing Tools and their Application in Experimental Ophthalmology

New genome editing tools in molecular biology are revolutionising precise genome surgery and have greatly influenced experimental ophthalmology too. Aside from the commonly used nuclease-based platforms, such as the zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), CRISPR/Cas systems, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, perform very efficiently in site-specific DNA cleavage within living cells. DNA double strand breaks (DSB) are repaired through two different conserved repair pathways: NHEJ (non-homologous end joining) and HDR (homology directed repair). By using the correct DNA templates, these repair pathways can be used to knock out defective genes or to repair mutations. Genome editing technology lays the ground for new strategies in basic science, biotechnology, and biomedical science, as well as clinical studies with genome editing. Therapeutic gene editing strategies are now concentrating on diseases in the retina, due to the comparatively easy accessibility of the eye and with local application in vivo.

Crystal structure of Pistol, a class of self-cleaving ribozyme

Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile by deprotonating the 2'-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5' leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.