Sequence-specific DNA Cleavage Research Articles

Hydrolytic endonucleolytic ribozyme (HYER) is programmable for sequence-specific DNA cleavage.

Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA endonucleases, which could be repurposed as DNA manipulation tools. Focused on bacterial group II-C introns, we found that many systems without intron-encoded protein propagate multiple copies in their resident genomes. These introns, named HYdrolytic Endonucleolytic Ribozymes (HYERs), cleaved RNA, single-stranded DNA, bubbled double-stranded DNA (dsDNA), and plasmids in vitro. HYER1 generated dsDNA breaks in the mammalian genome. Cryo-electron microscopy analysis revealed a homodimer structure for HYER1, where each monomer contains a Mg2+-dependent hydrolysis pocket and captures DNA complementary to the target recognition site (TRS). Rational designs including TRS extension, recruiting sequence insertion, and heterodimerization yielded engineered HYERs showing improved specificity and flexibility for DNA manipulation.

In-silicon studies on hydration in EcoRI-cognate DNA complex

Restriction endonucleases (REs) cleave DNA at specific site in presence of Mg2+ ion. Experiments further emphasize the role of hydration in metal ion specificity and sequence specificity of DNA cleavage. However, the relation between hydration and specificity has not been understood till date. This leads us to study via all-atom molecular dynamics (MD) simulations how the hydration around the scissile phosphate group changes in presence of Mg2+ and Ca2+ and depend on the DNA sequence. We observe the least number of hydrogen bonds around the scissile phosphate group in presence of Mg2+ ion. We further find that the hydrogen bonds decrease at the scissile phosphate on mutating one base pair in the cleavage region of the DNA in Mg2+ loaded EcoRI-DNA complex. We also perform steered MD simulations and observe that the rate of decrease of fraction of hydrogen bonds is slower in the mutated complex than the unmutated complex.

Folate Receptor-Mediated Delivery of Cas9 RNP for Enhanced Immune Checkpoint Disruption in Cancer Cells.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associatedprotein9(Cas9)system offers great opportunities for the treatment of numerous diseases by precise modification of the genome. The functional unit of the system is represented by Cas9/sgRNA ribonucleoproteins (RNP), which mediate sequence-specific cleavage of DNA. For therapeutic applications, efficient and cell-specific transport into target cells is essential. Here, Cas9 RNP nanocarriers are described, which are based on lipid-modified oligoamino amides and folic acid (FolA)-PEG to realize receptor-mediated uptake and gene editing in cancer cells. In vitro studies confirm strongly enhanced potency of receptor-mediated delivery, and the nanocarriers enable efficient knockout of GFP and two immune checkpoint genes, PD-L1 and PVR, at low nanomolar concentrations. Compared with non-targeted nanoparticles, FolA-modified nanocarriers achieve substantially higher gene editing including dual PD-L1/PVR gene disruption after injection into CT26 tumors in vivo. In the syngeneic mouse model, dual disruption of PD-L1 and PVR leads to CD8+ T cell recruitment and distinct CT26 tumor growth inhibition, clearly superior to the individual knockouts alone. The reported Cas9 RNP nanocarriers represent a versatile platform for potent and receptor-specific gene editing. In addition, the study demonstrates a promising strategy for cancer immunotherapy by permanent and combined immune checkpoint disruption.

Front Cover: A Simple Colorimetric Assay of Bleomycin‐Mediated DNA Cleavage Utilizing Double‐Stranded DNA‐Modified Gold Nanoparticles (ChemBioChem 1/2023)

A colorimetric assay of sequence-specific DNA cleavage by bleomycin (BLM) derivatives is a promising methodology for facilitating the development of DNA-associated drugs with antitumor activity. Double-stranded DNA-functionalized gold nanoparticles (dsDNA-AuNPs) possessing BLM-mediated DNA cleavage sites were designed. The front cover highlights a non-fluorescence-based analytical method that allows visual screening of potent antitumor agents using the DNA-protruding structure formed by BLM-mediated DNA cleavage on the outermost surfaces of the dsDNA-AuNPs. This study could provide a facile method for efficiently screening BLM-like drug candidates obtained from combinatorial synthesis. More information can be found in the Research Article by Y. Akiyama et al.

Harnessing CRISPR-Cas system diversity for gene editing technologies.

The discovery and utilization of RNA-guided surveillance complexes, such as CRISPR-Cas9, for sequence-specific DNA or RNA cleavage, has revolutionised the process of gene modification or knockdown. To optimise the use of this technology, an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements, coupled with high cleavage efficacy and specificity. Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.

Oxidative DNA Cleavage with Clip-Phenanthroline Triplex-Forming Oligonucleotide Hybrids.

A systematic study of several new types of hybrids of Cu-chelated clamped phenanthroline artificial metallonuclease (AMN) with triplex-forming oligonucleotides (TFO) for sequence-specific cleavage of double-stranded DNA (dsDNA) is reported. The synthesis of these AMN-TFO hybrids is based on application of the alkyne-azide cycloaddition click reaction as the key step. The AMN was attached through different linkers at either the 5'- or 3'-ends or in the middle of the TFO stretch. The diverse hybrids efficiently formed triplexes with the target purine-rich sequence and their copper complexes were studied for their ability to cleave dsDNA in the presence of ascorbate as a reductant. In all cases, the influence of the nature and length of the AMN-TFO, time, conditions and amounts of ascorbate were studied, and optimum conjugates and a procedure that gave reasonably efficient (up to 34 %) cleavage of the target sequence, while rendering an off-target dsDNA intact, were found. The footprint of cleavage on PAGE was identified only in one case, with low conversion; this means that cleavage does not proceed with single nucleotide precision. On the other hand, these AMN-TFO hybrids are useful for the selective degradation of target dsDNA sequences. Future improvements to this design may provide higher resolution and selectivity.

The genome-wide sequence specificity of DNA cleavage by bleomycin analogues in human cells

Bleomycin (BLM) is a cancer chemotherapeutic agent that cleaves cellular DNA at specific sequences. Using next-generation Illumina sequencing, the genome-wide sequence specificity of DNA cleavage by two BLM analogues, 6′-deoxy-BLM Z and zorbamycin (ZBM), was determined in human HeLa cells and compared with BLM. Over 200 million double-strand breaks were examined for each sample, and the 50,000 highest intensity cleavage sites were analysed. It was found that the DNA sequence specificity of the BLM analogues in human cells was different to BLM, especially at the cleavage site (position “0”) and the “+1” position. In human cells, the 6′-deoxy-BLM Z had a preference for 5′-GTGY*MC (where * is the cleavage site, Y is C or T, M is A or C); it was 5′-GTGY*MCA for ZBM; and 5′-GTGT*AC for BLM. With cellular DNA, the highest ranked tetranucleotides were 5′-TGC*C and 5′-TGT*A for 6′-deoxy-BLM Z; 5′-TGC*C, 5′-TGT*A and 5′-TGC*A for ZBM; and 5′-TGT*A for BLM. In purified human genomic DNA, the DNA sequence preference was 5′-TGT*A for 6′-deoxy-BLM, 5′-RTGY*AYR (where R is G or A) for ZBM, and 5′-TGT*A for BLM. Thus, the sequence specificity of the BLM analogue, 6′-deoxy-BLM Z, was similar to BLM in purified human DNA, while ZBM was different.

Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters.

CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct tissue/cell specific gene editing.

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems.

Adaptive immunity had been long thought of as an exclusive feature of animals. However, the discovery of the CRISPR-Cas defense system, present in almost half of prokaryotic genomes, proves otherwise. Because of the everlasting parasite-host arms race, CRISPR-Cas has rapidly evolved through horizontal transfer of complete loci or individual modules, resulting in extreme structural and functional diversity. CRISPR-Cas systems are divided into two distinct classes that each consist of three types and multiple subtypes. We discuss recent advances in CRISPR-Cas research that reveal elaborate molecular mechanisms and provide for a plausible scenario of CRISPR-Cas evolution. We also briefly describe the latest developments of a wide range of CRISPR-based applications.

Internal guide RNA interactions interfere with Cas9-mediated cleavage.

The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9–gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9–gRNA complex formation.

331. CRISPR-Cas9 Can Inhibit HIV-1 Replication but Also Facilitates Virus Escape

Several recent studies demonstrated that the CRISPR-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide insertions, deletions and substitutions around the Cas9/gRNA cleavage site that are typical for DNA repair by the NHEJ pathway. We thus demonstrate the potency of CRISPR-Cas9 as antiviral approach, but any therapeutic strategy should consider the viral escape options.

CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape

Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide insertions, deletions, and substitutions around the Cas9/gRNA cleavage site that are typical for DNA repair by the nonhomologous end-joining pathway. We thus demonstrate the potency of CRISPR-Cas9 as an antiviral approach, but any therapeutic strategy should consider the viral escape implications.

Bis((1,4-dimethyl-9H-carbazol-3-yl)methyl)amine-mediated anticancer effect triggered by sequence-specific cleavage of DNA leading to programmed cell death in the human U87 cell line

Bis((1,4-dimethyl-9H-carbazol-3-yl)methyl)amine (DMCM) has been designed as a novel anti-cancer agent which is believed to have a sequence-specific groove binding interaction with DNA, leading to anticancer effects.

A single molecule assay for measuring site-specific DNA cleavage

Sequence-specific DNA cleavage is a key step in a number of genomic transactions. Here, we report a single-molecule technique that allows the simultaneous measurement of hundreds of DNAs, thereby collecting significant statistics in a single experiment. Microbeads are tethered with single DNA molecules in a microfluidic channel. After the DNA cleavage reaction is initiated, the time of cleavage of each DNA is recorded using video microscopy. We demonstrate the utility of our method by measuring the cleavage kinetics of NdeI, a type II restriction endonuclease.

Programmable RNA recognition and cleavage by CRISPR/Cas9.

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to identify target sites for sequence-specific doublestranded DNA (dsDNA) cleavage1-5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, the protospacer adjacent motif (PAM), next to and on the strand opposite the 20-nucleotide target site in dsDNA4-7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in many cell types and organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous mRNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable and tagless transcript recognition.

Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing.

CRISPR/Cas9 uses a guide RNA (gRNA) molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.

Newer Gene Editing Technologies toward HIV Gene Therapy

Despite the great success of highly active antiretroviral therapy (HAART) in ameliorating the course of HIV infection, alternative therapeutic approaches are being pursued because of practical problems associated with life-long therapy. The eradication of HIV in the so-called “Berlin patient” who received a bone marrow transplant from a CCR5-negative donor has rekindled interest in genome engineering strategies to achieve the same effect. Precise gene editing within the cells is now a realistic possibility with recent advances in understanding the DNA repair mechanisms, DNA interaction with transcription factors and bacterial defense mechanisms. Within the past few years, four novel technologies have emerged that can be engineered for recognition of specific DNA target sequences to enable site-specific gene editing: Homing Endonuclease, ZFN, TALEN, and CRISPR/Cas9 system. The most recent CRISPR/Cas9 system uses a short stretch of complementary RNA bound to Cas9 nuclease to recognize and cleave target DNA, as opposed to the previous technologies that use DNA binding motifs of either zinc finger proteins or transcription activator-like effector molecules fused to an endonuclease to mediate sequence-specific DNA cleavage. Unlike RNA interference, which requires the continued presence of effector moieties to maintain gene silencing, the newer technologies allow permanent disruption of the targeted gene after a single treatment. Here, we review the applications, limitations and future prospects of novel gene-editing strategies for use as HIV therapy.

Towards Chemical Control of Gene Expression. Copper(II) and Thiols, Including Glutathione, are Powerful Reagents for Cleavage of DNA

Sequence-specific cleavage of DNA has many applications in molecular biology, but is limited by the specificities and accessibility of natural restriction endonucleases1. One approach to overcoming this has been to chemically modify a DNA-recognising molecule with a reagent capable of chemical cleavage of DNA. The most commonly used reagent for such chemical cleavage is probably the EDTA:Fe (II) system (1) which has been attached to oligonucleotides1,2, intercalators3, and to a combination of these binding-species4. Minor-groove directed drugs5 and antisense oligodeoxyribonucleoside methylphosphonates6 have joined the catalogue. The other commonly used chemical cleavage systems include bis (1,10- phenanthroline) :Cu (I) (2)7–9 porphyrin metal complexes (3)10–13, and rhodium complexes14. Photochemical cleaving systems for DNA have also been described15–19.

Sequence-Specific Ultrasonic Cleavage of DNA

We investigated the phenomenon of ultrasonic cleavage of DNA by analyzing a large set of cleavage patterns of DNA restriction fragments using polyacrylamide gel electrophoresis. The cleavage intensity of individual phosphodiester bonds was found to depend on the nucleotide sequence and the position of the bond with respect to the ends of the fragment. The relative intensities of cleavage of the central phosphodiester bond in 16 dinucleotides and 256 tetranucleotides were determined by multivariate statistical analysis. We observed a remarkable enhancement of the mean values of the relative intensities of cleavage (cleavage rates) in phosphodiester bonds following deoxycytidine, which diminished in the row of dinucleotides: d(CpG) > d(CpA) > d(CpT) >> d(CpC). The cleavage rates for all pairs of complementary dinucleotides were significantly different from each other. The effect of flanking nucleotides in tetranucleotides on cleavage rates of all 16 types of central dinucleotides was also statistically significant. The sequence-dependent ultrasonic cleavage rates of dinucleotides are consistent with reported data on the intensity of the conformational motion of their 5′-deoxyribose. As a measure of local conformational dynamics, cleavage rates may be useful for characterizing functional regions of the genome.

Sequence-specific DNA cleavage mediated by bipyridine polyamide conjugates

The design of molecules that damage a selected DNA sequence provides a formidable opportunity for basic and applied biology. For example, such molecules offer new prospects for controlled manipulation of the genome. The conjugation of DNA-code reading molecules such as polyamides to reagents that induce DNA damages provides an approach to reach this goal. In this work, we showed that a bipyridine conjugate of polyamides was able to induce sequence-specific DNA breaks in cells. We synthesized compounds based on two polyamide parts linked to bipyridine at different positions. Bipyridine conjugates of polyamides were found to have a high affinity for the DNA target and one of them produced a specific and high-yield cleavage in vitro and in cultured cells. The bipyridine conjugate studied here, also presents cell penetrating properties since it is active when directly added to cell culture medium. Harnessing DNA damaging molecules such as bipyridine to predetermined genomic sites, as achieved here, provides an attractive strategy for targeted genome modification and DNA repair studies.