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Current Opinion in Microbiology Jun 2017Adaptive immune systems in bacteria and archaea rely on small CRISPR-derived RNAs (crRNAs) to guide specialized nucleases to foreign nucleic acids. The activation of... (Review)
Review
Adaptive immune systems in bacteria and archaea rely on small CRISPR-derived RNAs (crRNAs) to guide specialized nucleases to foreign nucleic acids. The activation of these nucleases is controlled by a series of molecular checkpoints that ensure precise cleavage of nucleic acid targets, while minimizing toxic off-target cleavage events. In this review, we highlight recent advances in understanding regulatory mechanisms responsible for controlling the activation of these nucleases and identify emerging regulatory themes conserved across diverse CRISPR systems.
Topics: Allosteric Regulation; Archaea; Bacteria; CRISPR-Cas Systems; Deoxyribonucleases; Models, Biological
PubMed: 28646675
DOI: 10.1016/j.mib.2017.05.010 -
Methods (San Diego, Calif.) Nov 2018The rapid growth of the field of gene editing can largely be attributed to the discovery and optimization of designer endonucleases. These include zinc finger nucleases... (Review)
Review
The rapid growth of the field of gene editing can largely be attributed to the discovery and optimization of designer endonucleases. These include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regular interspersed short palindromic repeat (CRISPR) systems including Cas9, Cas12a, and structure-guided nucleases. Zebrafish (Danio rerio) have proven to be a powerful model system for genome engineering testing and applications due to their external development, high fecundity, and ease of housing. As the zebrafish gene editing toolkit continues to grow, it is becoming increasingly important to understand when and how to utilize which of these technologies for maximum efficacy in a particular project. While CRISPR-Cas9 has brought broad attention to the field of genome engineering in recent years, designer endonucleases have been utilized in genome engineering for more than two decades. This chapter provides a brief overview of designer endonuclease and other gene editing technologies in zebrafish as well as some of their known functional benefits and limitations depending on specific project goals. Finally, selected prospects for additional gene editing tools are presented, promising additional options for directed genomic programming of this versatile animal model system.
Topics: Animals; CRISPR-Cas Systems; DNA Repair; Deoxyribonucleases; Gene Editing; Genome; Protein Engineering; Zebrafish
PubMed: 30076892
DOI: 10.1016/j.ymeth.2018.07.012 -
Advanced Science (Weinheim,... Sep 2023Acute lung injury (ALI) is a frequent and serious complication of sepsis with limited therapeutic options. Gaining insights into the inflammatory dysregulation that...
Acute lung injury (ALI) is a frequent and serious complication of sepsis with limited therapeutic options. Gaining insights into the inflammatory dysregulation that causes sepsis-associated ALI can help develop new therapeutic strategies. Herein, the crucial role of cell-free mitochondrial DNA (cf-mtDNA) in the regulation of alveolar macrophage activation during sepsis-associated ALI is identified. Most importantly, a biocompatible hybrid protein nanomotor (NM) composed of recombinant deoxyribonuclease I (DNase-I) and human serum albumin (HSA) via glutaraldehyde-mediated crosslinking is prepared to obtain an inhalable nanotherapeutic platform targeting pulmonary cf-mtDNA clearance. The synthesized DNase-I/HSA NMs are endowed with self-propulsive capability and demonstrate superior performances in stability, DNA hydrolysis, and biosafety. Pulmonary delivery of DNase-I/HSA NMs effectively eliminates cf-mtDNAs in the lungs, and also improves sepsis survival by attenuating pulmonary inflammation and lung injury. Therefore, pulmonary cf-mtDNA clearance strategy using DNase-I/HSA NMs is considered to be an attractive approach for sepsis-associated ALI.
Topics: Humans; DNA, Mitochondrial; Acute Lung Injury; Lung; Sepsis; Deoxyribonucleases
PubMed: 37518854
DOI: 10.1002/advs.202301635 -
Advanced Science (Weinheim,... Nov 2023Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are prevalent critical illnesses with a high mortality rate among patients in intensive care units....
Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are prevalent critical illnesses with a high mortality rate among patients in intensive care units. Neutrophil extracellular traps (NETs) are implicated in the pathogenesis of ALI/ARDS and represent a promising therapeutic target. However, the clinical application of deoxyribonuclease I (DNase I), the only drug currently available to clear NETs, is limited due to the lack of precise and efficient delivery strategies. Therefore, targeted delivery of DNase I to the inflamed lung remains a critical issue to be addressed. Herein, a novel biomimetic DNase I delivery system is developed (DCNV) that employs genetically and bioorthogonally engineered cellular nanovesicles for pulmonary NETs clearance. The CXC motif chemokine receptor 2 overexpressed cellular nanovesicles can mimic the inflammatory chemotaxis of neutrophils in ALI/ARDS, leading to enhanced lung accumulation. Furthermore, DNase I immobilized through bioorthogonal chemistry exhibits remarkable enzymatic activity in NETs degradation, thus restraining inflammation and safeguarding lung tissue in the lipopolysaccharide-induced ALI murine model. Collectively, the findings present a groundbreaking proof-of-concept in the utilization of biomimetic cellular nanovesicles to deliver DNase I for treating ALI/ARDS. This innovative strategy may usher in a new era in the development of pharmacological interventions for various inflammation-related diseases.
Topics: Humans; Animals; Mice; Extracellular Traps; Acute Lung Injury; Respiratory Distress Syndrome; Inflammation; Deoxyribonuclease I
PubMed: 37759381
DOI: 10.1002/advs.202303053 -
Protein & Cell Dec 2016
Topics: Animals; Archaeal Proteins; Deoxyribonuclease I; Gene Editing; Humans; Natronobacterium
PubMed: 27848216
DOI: 10.1007/s13238-016-0343-9 -
Arteriosclerosis, Thrombosis, and... Sep 2020Macrophages are immune cells, capable to remodel the extracellular matrix, which can harbor extracellular DNA incorporated into neutrophil extracellular traps (NETs). To... (Comparative Study)
Comparative Study
OBJECTIVE
Macrophages are immune cells, capable to remodel the extracellular matrix, which can harbor extracellular DNA incorporated into neutrophil extracellular traps (NETs). To study the breakdown of NETs we studied the capability of macrophage subsets to degrade these structures in vitro and in vivo in a murine thrombosis model. Furthermore, we analyzed human abdominal aortic aneurysm samples in support of our in vitro and in vivo results. Approach and Results: Macrophages were seeded onto blood clots or isolated NETs and polarized. All macrophages were capable to degrade NETs. For initial breakdown, macrophages relied on extracellular deoxyribonucleases. Proinflammatory polarization enhanced NET degradation. The boost in degradation was because of increased macropinocytosis, as inhibition by imipramine diminished their NET breakdown. Inhibition of macropinocytosis in a murine thrombosis model led to increased NET burden and reduced thrombus resolution in vivo. When analyzing abdominal aortic aneurysm samples, macrophage density furthermore corresponded negatively with the amount of local NETs in the intraluminal thrombi as well as in the vessel wall, as increased macrophage density was associated with a reduction in NET burden.
CONCLUSIONS
We provide evidence that macrophages degrade NETs by extracellular predigestion and subsequent uptake. Furthermore, we show that proinflammatory macrophages increase NET degradation through enhanced macropinocytosis, priming them for NET engulfment. Based on our findings, that inhibition of macropinocytosis in mice corresponded to increased NET amounts in thrombi and that local macrophage density in human abdominal aortic aneurysm is negatively associated with surrounding NETs, we hypothesize, that macrophages are able to degrade NETs in vivo.
Topics: Animals; Aortic Aneurysm, Abdominal; Cells, Cultured; Deoxyribonuclease I; Deoxyribonucleases; Disease Models, Animal; Endodeoxyribonucleases; Exodeoxyribonucleases; Extracellular Traps; Female; Humans; Imipramine; Interferon-gamma; Interleukin-13; Interleukin-4; Kinetics; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; Muscle Proteins; Neutrophils; Phagocytosis; Phenotype; Phosphoproteins; Pinocytosis; Vena Cava, Inferior; Venous Thrombosis
PubMed: 32673525
DOI: 10.1161/ATVBAHA.120.314883 -
ELife Sep 2022The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell...
The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.
Topics: Humans; Gene Editing; Pluripotent Stem Cells; Deoxyribonuclease I; RNA, Messenger; RNA-Directed DNA Polymerase; Ribonucleoproteins; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems
PubMed: 36069759
DOI: 10.7554/eLife.79208 -
Nucleic Acids Research Jun 2017Deoxyribonuclease II (DNase II) is also known as acid deoxyribonuclease because it has optimal activity at the low pH environment of lysosomes where it is typically... (Comparative Study)
Comparative Study
Deoxyribonuclease II (DNase II) is also known as acid deoxyribonuclease because it has optimal activity at the low pH environment of lysosomes where it is typically found in higher eukaryotes. Interestingly, DNase II has also been identified in a few genera of bacteria and is believed to have arisen via horizontal transfer. Here, we demonstrate that recombinant Burkholderia thailandensis DNase II is highly active at low pH in the absence of divalent metal ions, similar to eukaryotic DNase II. The crystal structure of B. thailandensis DNase II shows a dimeric quaternary structure which appears capable of binding double-stranded DNA. Each monomer of B. thailandensis DNase II exhibits a similar overall fold as phospholipase D (PLD), phosphatidylserine synthase (PSS) and tyrosyl-DNA phosphodiesterase (TDP), and conserved catalytic residues imply a similar mechanism. The structural and biochemical data presented here provide insights into the atomic structure and catalytic mechanism of DNase II.
Topics: Amino Acid Sequence; Bacterial Proteins; Burkholderia; Catalytic Domain; Copper; Crystallography, X-Ray; DNA, Bacterial; Endodeoxyribonucleases; Eukaryotic Cells; Evolution, Molecular; Gene Transfer, Horizontal; Hydrogen-Ion Concentration; Models, Molecular; Molecular Docking Simulation; Phylogeny; Prokaryotic Cells; Protein Conformation; Protein Folding; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid
PubMed: 28369538
DOI: 10.1093/nar/gkx222 -
JAMA Network Open Apr 2023There is a paucity of high-quality prospective randomized clinical trials comparing intrapleural fibrinolytic therapy (IPFT) with surgical decortication in patients with... (Randomized Controlled Trial)
Randomized Controlled Trial
IMPORTANCE
There is a paucity of high-quality prospective randomized clinical trials comparing intrapleural fibrinolytic therapy (IPFT) with surgical decortication in patients with complicated pleural infections.
OBJECTIVE
To assess the feasibility, safety, and efficacy of an algorithm comparing tissue plasminogen activator plus deoxyribonuclease therapy with surgical decortication in patients with complicated pleural infections.
DESIGN, SETTING, AND PARTICIPANTS
This parallel pilot randomized clinical trial was performed at a single urban community-based center from March 1, 2019, to December 31, 2021, with follow-up for 90 days. Seventy-four individuals were screened and 48 were excluded. Twenty-six patients 18 years or older with clinical pleural infection and positive findings of pleural fluid analysis were included. Of these, 20 patients underwent randomized selection (10 in each group), and 6 were observed.
INTERVENTIONS
Intrapleural tissue plasminogen activator plus deoxyribonuclease therapy vs surgical decortication.
MAIN OUTCOMES AND MEASURES
Primary outcomes were the percentage of patients enrolled to study completion and multidisciplinary adherence. Secondary outcomes included the number of patients with and the reason for inadequate screening, screening to enrollment failures, time to accrual of 20 patients or the number accrued at 1 year, and clinical data.
RESULTS
Twenty-six patients were enrolled, 10 were randomized to each group, and 6 were observed. There was 100% enrollment to study completion in each treatment group, no protocol deviations, 2 minor protocol amendments, and no screening to enrollment failures. It took 32 months to enroll 26 patients. The 20 randomized patients had a median age of 57 (IQR, 46-65) years, were predominantly men (15 [75%]), and had a median RAPID (Renal, Age, Purulence, Infection Source, and Dietary Factors) score of 2 (IQR, 1-3). Treatment failure occurred in 1 patient and 2 crossover treatments occurred, all of which were in the IPFT group. Intraprocedure and postprocedure complications were similar between the groups. There were no reoperations or in-hospital deaths. Median duration of chest tube use was comparable in the IPFT (5 [IQR, 4-8] days) and surgery (4 [IQR, 3-5] days) groups (P = .21). Median hospital stay tended to be longer in the IPFT (11 [IQR, 4-18] days) vs surgery (5 [IQR, 4-6] days) groups, although the difference as not significantly different (P = .08). There were no 30-day readmissions or 30- or 90-day deaths.
CONCLUSIONS AND RELEVANCE
In this pilot randomized clinical trial, the study algorithm was feasible, safe, and efficacious. This provides evidence to move forward with a multicenter randomized clinical trial.
TRIAL REGISTRATION
ClinicalTrials.gov Identifier: NCT03873766.
Topics: Male; Humans; Middle Aged; Aged; Female; Tissue Plasminogen Activator; Fibrinolytic Agents; Prospective Studies; Thrombolytic Therapy; Communicable Diseases; Deoxyribonucleases
PubMed: 37043201
DOI: 10.1001/jamanetworkopen.2023.7799 -
Nature Medicine Feb 2015Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the... (Review)
Review
Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease-based therapies as well as future prospects and challenges.
Topics: Deoxyribonucleases; Endodeoxyribonucleases; Genetic Engineering; Genetic Therapy; Genome; Humans
PubMed: 25654603
DOI: 10.1038/nm.3793