Despite the extensive and rapid discovery of modern drugs for treatment of cancer, microbial infections, and viral illnesses; these diseases are still among major global health concerns. To take inspiration from natural nucleases and also the therapeutic potential of metallopeptide antibiotics such as the bleomycin family, artificial metallonucleases with the ability of promoting DNA/RNA cleavage and eventually affecting cellular biological processes can be introduced as a new class of therapeutic candidates. Metal complexes can be considered as one of the main categories of artificial metalloscissors, which can prompt nucleic acid strand scission.
Accordingly, biologists, inorganic chemists, and medicinal inorganic chemists worldwide have been designing, synthesizing and evaluating the biological properties of metal complexes as artificial metalloscissors. In this review, we try to highlight the recent studies conducted on the nuclease-like metalloscissors and their potential therapeutic applications. Under the light of the concurrent Covid-19 pandemic, the human need for new therapeutics was highlighted much more than ever before. The nuclease-like metalloscissors with the potential of RNA cleavage of invading viral pathogens hence deserve prime attention.
Mechanistic insights of CRISPR/Cas nucleases for programmable targeting and early-stage diagnosis: A review
- Conventional and routine diagnostics such as polymerase chain reaction (PCR) and serological tests are less sensitive, costly, and require sample pretreatment procedures. CRISPR/Cas systems that inherently assist bacteria and archaea in destroying invading phage genetic materials via an RNA-mediated interference strategy have been reconstituted in vitro and harnessed for nucleic and non-nucleic acid diagnostics. CRISPR/Cas-based diagnostics (CRISPR-Dx) are cost-effective, possess excellent sensitivity (attomolar) and specificity (single base distinction), exhibit fast turnaround response, and support nucleic acid extraction-free workflow.
- However, CRISPR-Dx still needs to address various challenges to translate the laboratory work into end-user tailored solutions. In this perspective, we review the relevant progress of CRISPR/Cas systems-based diagnostics, focusing on the comprehensive customization and applications of leading and trending CRISPR/Cas systems as platform technologies for fluorescence, colorimetric, and electrical signal detection. The impact of the CRISPR game-changing technology on the COVID-19 pandemic is highlighted. We also demonstrate the role of CRISPR/Cas systems for carryover contamination prevention.
- The advancements in signal amplification strategies using engineered crRNAs, novel reporters, nanoparticles, artificial genetic circuits, microfluidics, and smartphones are also covered. Furthermore, we critically discuss the translation of CRISPR-Dx’s basic research into end-user diagnostics for commercialization success in the near future. Finally, we discuss the complex challenges and alternative solutions to harness the CRISPR/Cas potential in detail.
Micrococcal Nuclease stimulates Staphylococcus aureus Biofilm Formation in a Murine Implant Infection Model
- Advancements in contemporary medicine have led to an increasing life expectancy which has broadened the application of biomaterial implants. As each implant procedure has an innate risk of infection, the number of biomaterial-associated infections keeps rising. Staphylococcus aureus causes 34% of such infections and is known as a potent biofilm producer. By secreting micrococcal nuclease S. aureus is able to escape neutrophil extracellular traps by cleaving their DNA-backbone.
- Also, micrococcal nuclease potentially limits biofilm growth and adhesion by cleaving extracellular DNA, an important constituent of biofilms. This study aimed to evaluate the impact of micrococcal nuclease on infection persistence and biofilm formation in a murine biomaterial-associated infection-model with polyvinylidene-fluoride mesh implants inoculated with bioluminescent S. aureus or its isogenic micrococcal nuclease deficient mutant. Supported by results based on in-vivo bioluminescence imaging, ex-vivo colony forming unit counts, and histological analysis it was found that production of micrococcal nuclease enables S. aureus bacteria to evade the immune response around an implant resulting in a persistent infection.
- As a novel finding, histological analysis provided clear indications that the production of micrococcal nuclease stimulates S. aureus to form biofilms, the presence of which extended neutrophil extracellular trap formation up to 13 days after mesh implantation. Since micrococcal nuclease production appeared vital for the persistence of S. aureus biomaterial-associated infection, targeting its production could be a novel strategy in preventing biomaterial-associated infection.
S1 Nuclease |
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E1335-01 | EURx | 10000U | 43.6 EUR |
S1 Nuclease |
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E1335-02 | EURx | 50000U | 168.95 EUR |
Cas9 Nuclease |
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314HC | GeneOn | 400 µg (2500 pmol) | 894 EUR |
Cas9 Nuclease |
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310 | GeneOn | 2x40µg (2x250 pmol) | 316.8 EUR |
Nuclease AWAY |
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A6257-100ML | Biomatik Corporation | 100ML | 22 EUR |
Nuclease AWAY |
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A6257-500ML | Biomatik Corporation | 500ML | 88 EUR |
Cas9 Nuclease |
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312HC | GeneOn | 80 µg (500 pmol) | 268.8 EUR |
Cas9 Nuclease |
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3276 | Intact Genomics | 400µg(1600ng/µl),2500pmol | 396 EUR |
Cas9 Nuclease |
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3273 | Intact Genomics | 80µg(1600ng/µl),500pmol | 107.2 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 |
OMNI Nuclease |
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E1120-01 | EURx | 20000U | 35.97 EUR |
OMNI Nuclease |
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E1120-02 | EURx | 100000U | 162.41 EUR |
Turbo Nuclease |
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EN-180L | Jena Bioscience GmbH | 5 x 10000units | 290.68 EUR |
Turbo Nuclease |
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EN-180S | Jena Bioscience GmbH | 10000units | 72.73 EUR |
Ultra Nuclease |
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HBP000106 | HZymes Biotechnology | 20μL | 14.73 EUR |
Ultra Nuclease |
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HBP000107 | HZymes Biotechnology | 200μL | 116.82 EUR |
Ultra Nuclease |
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HBP000108 | HZymes Biotechnology | 2mL | 965.04 EUR |
Ultra Nuclease |
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HBP000109 | HZymes Biotechnology | 20mL | 8634.56 EUR |
Cas12a Nuclease |
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3373 | Intact Genomics | 80µg(1600ng/µl) | 123.2 EUR |
Cas12a Nuclease |
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3376 | Intact Genomics | 400µg(1600ng/µl) | 420 EUR |
Nuclease P1 (Nuclease 5'-Nucleotidehydrolase, 3'-Phosphohydrolase, NP1) |
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MBS653730-100Units | MyBiosource | 100Units | 320 EUR |
Nuclease P1 (Nuclease 5'-Nucleotidehydrolase, 3'-Phosphohydrolase, NP1) |
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MBS653730-250Units | MyBiosource | 250Units | 635 EUR |
Nuclease P1 (Nuclease 5'-Nucleotidehydrolase, 3'-Phosphohydrolase, NP1) |
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MBS653730-5x250Units | MyBiosource | 5x250Units | 2710 EUR |
Benzonase Nuclease |
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MBS157420-100KUnits | MyBiosource | 100KUnits | 320 EUR |
Benzonase Nuclease |
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MBS157420-25KUnits | MyBiosource | 25KUnits | 210 EUR |
Targeted deletion of glycoprotein B gene by CRISPR/Cas9 nuclease inhibits Gallid herpesvirus type 3 in dually-infected Marek’s disease virus-transformed lymphoblastoid cell line MSB-1
Marek’s disease virus (MDV) is a member of the genus Mardivirus in the subfamily Alphaherpesvirinae. There are 3 different serotypes of MDV designated as MDV-1 (Gallid herpesvirus type 2), MDV-2 (Gallid herpesvirus type 3), and MDV-3 (Meleagrid herpesvirus 1, herpesvirus of turkeys, HVT). MDV-1 is the only serotype that induces Marek’s disease (MD), a lymphoproliferative disorder resulting in aggressive T-cell lymphomas and paralytic symptoms. In the lymphomas and lymphoblastoid cell lines (LCL) derived from them, MDV establishes latent infection with limited viral gene expression.
The latent viral genome in LCL can be activated by co-cultivation with chicken embryo fibroblast (CEF) monolayers. MSB-1, one of the first MDV-transformed LCL established from the splenic lymphoma, is distinct in harbouring both the oncogenic MDV-1 and non-oncogenic MDV-2 viruses. Following the successful application of CRISPR/Cas9 editing approach for precise knockdown of the MDV-1 genes in LCL, we describe here the targeted deletion of MDV-2 glycoprotein B (gB) in MSB-1 cells. Due to the essential nature of gB for infectivity, the production of MDV-2 plaques on CEF was completely abolished in the MDV-2-gB-deleted MSB-1 cells. Our study has demonstrated that the CRISPR/Cas9 system can be used for targeted inactivation of the co-infecting MDV-2 without affecting the MDV-1 in the MSB-1 cell line.
Successful inactivation of MDV-2 demonstrated here also points towards the possibility of using targeted gene editing as an antiviral strategy against pathogenic MDV-1 and other viruses infecting chickens. Importance Marek’s disease (MD) is a lymphoproliferative disease of chickens characterized by rapid-onset lymphomas in multiple organs and by infiltration into peripheral nerves, causing paralysis. Lymphoblastoid cell lines (LCL) derived from MD lymphomas have served as valuable resources to improve understanding of distinct aspects of virus-host interactions in transformed cells including transformation, latency and reactivation. MDV-transformed LCL MSB-1, derived from spleen lymphoma induced by the BC-1 strain of MDV, has a unique feature of harbouring an additional non-pathogenic MDV-2 strain HPRS-24. By targeted deletion of essential gene glycoprotein B from the MDV-2 genome within the MSB-1 cells, we demonstrated the total inhibition of MDV-2 virus replication on co-cultivated CEF, with no effect on MDV-1 replication. The identified viral genes critical for reactivation/inhibition of viruses will be useful as targets for development of de novo disease resistance in chickens to avian pathogens.