- Methylcytosines in mammalian genomes are the main epigenetic molecular codes that switch off the repertoire of genes in cell-type and cell-stage dependent manners. DNA methyltransferases (DMT) are dedicated to managing the status of cytosine methylation. DNA methylation is not only critical in normal development, but it is also implicated in cancers, degeneration, and senescence. Thus, the chemicals to control DMT have been suggested as anticancer drugs by reprogramming the gene expression profile in malignant cells.
- Here, we report a new optical technique to characterize the activity of DMT and the effect of inhibitors, utilizing the methylation-sensitive B-Z transition of DNA without bisulfite conversion, methylation-sensing proteins, and polymerase chain reaction amplification. With the high sensitivity of single-molecule FRET, this method detects the event of DNA methylation in a single DNA molecule and circumvents the need for amplification steps, permitting direct interpretation. This method also responds to hemi-methylated DNA.
- Dispensing with methylation-sensitive nucleases, this method preserves the molecular integrity and methylation state of target molecules. Sparing methylation-sensing nucleases and antibodies helps to avoid errors introduced by the antibody’s incomplete specificity or variable activity of nucleases. With this new method, we demonstrated the inhibitory effect of several natural bio-active compounds on DMT. All taken together, our method offers quantitative assays for DMT and DMT-related anticancer drugs.
Rapidly Characterizing CRISPR-Cas13 Nucleases Using Cell-Free Transcription-Translation Systems
Cell-free transcription-translation (TXTL) systems produce RNAs and proteins from added DNA. By coupling their production to a biochemical assay, these biomolecules can be rapidly and scalably characterized without the need for purification or cell culturing. Here, we describe how TXTL can be applied to characterize Cas13 nucleases from Type VI CRISPR-Cas systems.
These nucleases employ guide RNAs to recognize complementary RNA targets, leading to the nonspecific collateral cleavage of nearby RNAs. In turn, RNA targeting by Cas13 has been exploited for numerous applications, including in vitro diagnostics, programmable gene silencing in eukaryotes, and sequence-specific antimicrobials. As part of the described method, we detail how to set up TXTL assays to measure on-target and collateral RNA cleavage by Cas13 as well as how to assay for putative anti-CRISPR proteins. Overall, the method should be useful for the characterization of Type VI CRISPR-Cas systems and their use in ranging applications.
Label-free and sensitive MiRNA detection based on turn-on fluorescence of DNA-templated silver nanoclusters coupled with duplex-specific nuclease-assisted signal amplification
A novel strategy for microRNAs (miRNAs) detection has been developed utilizing duplex-specific nuclease-assisted signal amplification (DSNSA) and guanine-rich DNA-enhanced fluorescence of DNA-templated silver nanoclusters (AgNCs). The combination between target miRNA, DSNSA, and AgNCs is achieved by the unique design of DNA sequences. Target miRNA opens the hairpin structure of the Hairpin DNA probe (HP) by hybridizing with the HP and initiates the duplex-specific nuclease-assisted signal amplification (DSNSA) reaction.
The DSNSA reaction generates the release of the guanine-rich DNA sequence, which can turn on the fluorescence of the dark AgNCs by hybridizing with the DNA template of the dark AgNCs. The fluorescence intensity of AgNCs corresponds to the dosage of the target miRNA. This is measured at 630 nm by exciting at 560 nm. The constructed method exhibits a low detection limit (~8.3 fmol), a great dynamic range of more than three orders of magnitude, and excellent selectivity. Moreover, it has a good performance for miR-21 detection in complex biological samples. A novel strategy for microRNAs (miRNAs) detection has been developed utilizing duplex-specific nuclease-assisted signal amplification (DSNSA) and guanine-rich DNA-enhanced fluorescence of DNA-templated silver nanoclusters (AgNCs).
Nuclease deficiencies alter plasma cell-free DNA methylation profiles
The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation was explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in Dnase1l3-deficient mice was significantly hypomethylated, while cfDNA in Dnase1-deficient mice was hypermethylated. The cfDNA hypomethylation in Dnase1l3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs).
These findings were absent in Dnase1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends and coverage at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affected cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology and cfDNA fragmentation.
Human Free PSA (f-PSA) ELISA Kit |
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| PRB-5049-FREE | Cell Biolabs | 96 assays | 686.4 EUR |
Human Free PSA (f-PSA) ELISA Kit |
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| PRB-5049-FREE-5 | Cell Biolabs | 5 x 96 assays | 2739.6 EUR |
Cas9 Nuclease |
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| EN301-01 | Vazyme | 50 pmol | 146.4 EUR |
Cas9 Nuclease |
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| EN301-02 | Vazyme | 250 pmol | 184.8 EUR |
Cas9 Nuclease |
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| 310 | GeneOn | 2x40µg (2x250 pmol) | 316.8 EUR |
Cas9 Nuclease |
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| 312HC | GeneOn | 80 µg (500 pmol) | 268.8 EUR |
Cas9 Nuclease |
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| 314HC | GeneOn | 400 µg (2500 pmol) | 894 EUR |
BenzNuclease DNA and RNA Nuclease (Ultra Pure, Protease Free) Recombinant Protein |
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| 11-552 | ProSci | 50KU | 481.2 EUR |
Nuclease P1 Antibody |
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| 1-CSB-PA328459LA01PEU | Cusabio |
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Nuclease P1 Antibody |
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| 20-abx300888 | Abbexa |
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Nuclease Mix 0.5mL |
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| 80650142 | Scientific Laboratory Supplies | EACH | 147.06 EUR |
Penicillium citrinum Nuclease P1 |
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| 1-CSB-YP328459PEU | Cusabio |
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Penicillium citrinum Nuclease P1 |
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| 1-CSB-EP328459PEU | Cusabio |
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Penicillium citrinum Nuclease P1 |
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| 1-CSB-EP328459PEUa0 | Cusabio |
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Apoptosis enhancing nuclease antibody |
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| 70R-13466 | Fitzgerald | 100 ul | 548.4 EUR |
Apoptosis-enhancing nuclease antibody |
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| 22725-100ul | SAB | 100ul | 468 EUR |
Nuclease P1 Antibody (HRP) |
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| 20-abx300889 | Abbexa |
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Nuclease P1 Antibody (FITC) |
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| 20-abx300890 | Abbexa |
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Nuclease P1 Antibody (Biotin) |
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| 20-abx300891 | Abbexa |
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Cas9 Nuclease Lentiviral Vector |
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| K002 | ABM | 10 ug | 184.8 EUR |
anti-Apoptosis enhancing nuclease |
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| YF-PA20583 | Abfrontier | 50 ug | 435.6 EUR |
Nuclease P1 Polyclonal Antibody |
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| A64318-020 | EpiGentek | 20 ul | 117.7 EUR |
Nuclease P1 Polyclonal Antibody |
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| A64318-050 | EpiGentek | 50 ul | 302.5 EUR |
Nuclease P1 Polyclonal Antibody |
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| A64318-100 | EpiGentek | 100 ul | 423.5 EUR |
Nuclease P1 Polyclonal Antibody |
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| A64318 | EpiGentek |
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Nuclease P1 Antibody, HRP conjugated |
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| 1-CSB-PA328459LB01PEU | Cusabio |
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Nuclease P1 Antibody, FITC conjugated |
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| 1-CSB-PA328459LC01PEU | Cusabio |
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Nuclease P1 Antibody, Biotin conjugated |
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| 1-CSB-PA328459LD01PEU | Cusabio |
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CRISPR/Cas-Dependent and Nuclease–Free In Vivo Therapeutic Gene Editing
Precise gene manipulation by gene editing approaches facilitates the potential to cure several debilitating genetic disorders. Gene modification stimulated by engineered nucleases induces a double-stranded break (DSB) in the target genomic locus, thereby activating DNA repair mechanisms. DSBs triggered by nucleases are repaired either by the nonhomologous end-joining or the homology-directed repair pathway, enabling efficient gene editing. While there are several ongoing ex vivo genome editing clinical trials, current research underscores the therapeutic potential of CRISPR/Cas-based (clustered regularly interspaced short palindrome repeats-associated Cas nuclease) in vivo gene editing.
In this review, we provide an overview of the CRISPR/Cas-mediated in vivo genome therapy applications and explore their prospective clinical translatability to treat human monogenic disorders. In addition, we discuss the various challenges associated with in vivo genome editing technologies and strategies used to circumvent them. Despite the robust and precise nuclease-mediated gene editing, a promoterless, nuclease-independent gene targeting strategy has been utilized to evade the drawbacks of the nuclease-dependent system, such as off-target effects, immunogenicity, and cytotoxicity. Thus, the rapidly evolving paradigm of gene editing technologies will continue to foster the progress of gene therapy applications.