7,380 results on '"Nucleases"'
Search Results
202. Nuclease activity: an exploitable biomarker in bacterial infections.
- Author
-
Garcia Gonzalez, Javier and Hernandez, Frank J.
- Abstract
In the increasingly challenging field of clinical microbiology, diagnosis is a cornerstone whose accuracy and timing are crucial for the successful management, therapy, and outcome of infectious diseases. Currently employed biomarkers of infectious diseases define the scope and limitations of diagnostic techniques. As such, expanding the biomarker catalog is crucial to address unmet needs and bring about novel diagnostic functionalities and applications. This review describes the extracellular nucleases of 15 relevant bacterial pathogens and discusses the potential use of nuclease activity as a diagnostic biomarker. Articles were searched for in PubMed using the terms: 'nuclease,' 'bacteria,' 'nuclease activity' or 'biomarker.' For overview sections, original and review articles between 2000 and 2019 were searched for using the terms: 'infections,' 'diagnosis,' 'bacterial,' 'burden,' 'challenges.' Informative articles were selected. Using the catalytic activity of nucleases offers new possibilities compared to established biomarkers. Nucleic acid activatable reporters in combination with different transduction platforms and delivery methods can be used to detect disease-associated nuclease activity patterns in vitro and in vivo for prognostic and diagnostic applications. Even when these patterns are not obvious or of unknown etiology, screening platforms could be used to identify new disease reporters. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
203. Genomic Reporter Constructs to Monitor Pathway-Specific Repair of DNA Double-Strand Breaks.
- Author
-
van de Kooij, Bert and van Attikum, Haico
- Subjects
DOUBLE-strand DNA breaks ,DNA repair ,FLUORESCENT proteins ,REPAIRING ,GENOME editing ,PROTEIN expression ,NUCLEASES - Abstract
Repair of DNA Double-Strand Breaks (DSBs) can be error-free or highly mutagenic, depending on which of multiple mechanistically distinct pathways repairs the break. Hence, DSB-repair pathway choice directly affects genome integrity, and it is therefore of interest to understand the parameters that direct repair towards a specific pathway. This has been intensively studied using genomic reporter constructs, in which repair of a site-specific DSB by the pathway of interest generates a quantifiable phenotype, generally the expression of a fluorescent protein. The current developments in genome editing with targetable nucleases like Cas9 have increased reporter usage and accelerated the generation of novel reporter constructs. Considering these recent advances, this review will discuss and compare the available DSB-repair pathway reporters, provide essential considerations to guide reporter choice, and give an outlook on potential future developments. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
204. Diversity and function of arthropod endosymbiont toxins.
- Author
-
Massey, Jonathan H. and Newton, Irene L.G.
- Subjects
- *
ARTHROPOD diversity , *BACTERIAL toxins , *BACTERIAL proteins , *TOXINS , *GLYCOSYLTRANSFERASES , *NUCLEASES , *WOLBACHIA - Abstract
Bacterial endosymbionts induce dramatic phenotypes in their arthropod hosts, including cytoplasmic incompatibility, feminization, parthenogenesis, male killing, parasitoid defense, and pathogen blocking. The molecular mechanisms underlying these effects remain largely unknown but recent evidence suggests that protein toxins secreted by the endosymbionts play a role. Here, we describe the diversity and function of endosymbiont proteins with homology to known bacterial toxins. We focus on maternally transmitted endosymbionts belonging to the Wolbachia , Rickettsia , Arsenophonus , Hamiltonella , Spiroplasma , and Cardinium genera because of their ability to induce the above phenotypes. We identify at least 16 distinct toxin families with diverse enzymatic activities, including AMPylases, nucleases, proteases, and glycosyltransferases. Notably, several annotated toxins contain domains with homology to eukaryotic proteins, suggesting that arthropod endosymbionts mimic host biochemistry to manipulate host physiology, similar to bacterial pathogens. Arthropod endosymbiont genomes contain multiple proteins with homology to bacterial protein toxins. Among Wolbachia , Rickettsia , Arsenophonus , Hamiltonella , Spiroplasma , and Cardinium genera, we identify at least 16 distinct toxin families. Several toxins contain multiple domains, share homology with eukaryotic proteins, and are encoded by bacteriophage genomes. Toxins known to induce male killing, cytoplasmic incompatibility, or parasitoid defense target host DNA or RNA. Arthropod endosymbionts might induce many of their host phenotypes via toxin secretion. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
205. WITH CRISPR/CAS9, FIGHTING AGAINST THE DARK EMPIRE OF DISEASES.
- Author
-
Kulshrestha, Saksham, Chauhan, Shailandra, and Singh, Gaurav
- Subjects
ZINC-finger proteins ,GENOME editing ,CRISPRS ,ENDONUCLEASES ,NUCLEASES - Abstract
Clustered Regularly InterSpaced Palindromic Repeats is what CRISPR stands for. This is a protein that is programmable and can edit, eliminate, or turn the genome on or off. This is a modern technology that can have many potential applications and can change the future of oral health for the better. Genome editing technologies can be used to create mutations in the genomes based on a set of sequence-specific instructions, including, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPRassociated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and homing endonucleases or meganucleases. CRISPR-Cas9 genome editing technology is very young, but it has emerged in recent years as a powerful tool in cancer therapy for its high efficiency and accuracy. The CRISPR-Cas9 system uses gene editing in cells, organs and animals to access entire genomes within a short period of time, both gene-editing for gain-of-function and gene-editing for loss-of-function. Methods of detection based on CRISPR/Cas by the near future may become simpler, more affordable, and faster. Here, we review the application of CRISPR-Cas9 genome editing in dentistry, as well as its general advantages and disadvantages. [ABSTRACT FROM AUTHOR]
- Published
- 2022
206. CRISPR-based genome editing through the lens of DNA repair.
- Author
-
Nambiar, Tarun S., Baudrier, Lou, Billon, Pierre, and Ciccia, Alberto
- Subjects
- *
GENOME editing , *DNA repair , *CRISPRS , *DNA damage , *RECOMBINASES , *POLLUTION , *NUCLEASES - Abstract
Genome editing technologies operate by inducing site-specific DNA perturbations that are resolved by cellular DNA repair pathways. Products of genome editors include DNA breaks generated by CRISPR-associated nucleases, base modifications induced by base editors, DNA flaps created by prime editors, and integration intermediates formed by site-specific recombinases and transposases associated with CRISPR systems. Here, we discuss the cellular processes that repair CRISPR-generated DNA lesions and describe strategies to obtain desirable genomic changes through modulation of DNA repair pathways. Advances in our understanding of the DNA repair circuitry, in conjunction with the rapid development of innovative genome editing technologies, promise to greatly enhance our ability to improve food production, combat environmental pollution, develop cell-based therapies, and cure genetic and infectious diseases. Nambiar et al. review recently developed CRISPR-based technologies, with an emphasis on the DNA lesions generated by these technologies and the DNA repair processes that resolve them. Understanding the interplay between CRISPR-based genome editing and DNA repair will provide strategies to enhance editing efficiency, optimize editing outcomes, and improve safety. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
207. The CRISPR-Cas toolbox and gene editing technologies.
- Author
-
Liu, Guanwen, Lin, Qiupeng, Jin, Shuai, and Gao, Caixia
- Subjects
- *
GENOME editing , *GENE expression , *LIFE sciences , *NUCLEASES , *EPIGENETICS - Abstract
The emergence of CRISPR-Cas systems has accelerated the development of gene editing technologies, which are widely used in the life sciences. To improve the performance of these systems, workers have engineered and developed a variety of CRISPR-Cas tools with a broader range of targets, higher efficiency and specificity, and greater precision. Moreover, CRISPR-Cas-related technologies have also been expanded beyond making cuts in DNA by introducing functional elements that permit precise gene modification, control gene expression, make epigenetic changes, and so on. In this review, we introduce and summarize the characteristics and applications of different types of CRISPR-Cas tools. We discuss certain limitations of current approaches and future prospects for optimizing CRISPR-Cas systems. Liu et al. review the development of the CRISPR-Cas toolbox and gene editing technologies, including the Cas nucleases used for gene editing as well as CRISPR-Cas-mediated DSB-dependent and DSB-independent gene editing tools, with an emphasis on precision tools and CRISPR-Cas-related technologies. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
208. Single-strand specific nuclease enhances accuracy of error-corrected sequencing and improves rare mutation-detection sensitivity.
- Author
-
Otsubo, Yuki, Matsumura, Shoji, Ikeda, Naohiro, and Yamane, Masayuki
- Subjects
- *
MUNG bean , *DNA sequencing , *NUCLEASES , *MUTAGENS , *ANTHRACENE - Abstract
Error-corrected sequences (ECSs) that utilize double-stranded DNA sequences are useful in detecting mutagen-induced mutations. However, relatively higher frequencies of G:C > T:A (1 × 10−7 bp) and G:C > C:G (2 × 10−7 bp) errors decrease the accuracy of detection of rare G:C mutations (approximately 10−7 bp). Oxidized guanines in single-strand (SS) overhangs generated after shearing could serve as the source of these errors. To remove these errors, we first computationally discarded up to 20 read bases corresponding to the ends of the DNA fragments. Error frequencies decreased proportionately with trimming length; however, the results indicated that they were not sufficiently removed. To efficiently remove SS overhangs, we evaluated three mechanistically distinct SS-specific nucleases (S1 Nuclease, mung bean nuclease, and RecJf exonuclease) and found that they were more efficient than computational trimming. Consequently, we established Jade-Seq™, an ECS protocol with S1 Nuclease treatment, which reduced G:C > T:A and G:C > C:G errors to 0.50 × 10−7 bp and 0.12 × 10−7 bp, respectively. This was probably because S1 Nuclease removed SS regions, such as gaps and nicks, depending on its wide substrate specificity. Subsequently, we evaluated the mutation-detection sensitivity of Jade-Seq™ using DNA samples from TA100 cells exposed to 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene, which contained the rare G:C > T:A mutation (i.e., 2 × 10−7 bp). Fold changes of G:C > T:A compared to the vehicle control were 1.2- and 1.3-times higher than those of samples without S1 Nuclease treatment, respectively. These findings indicate the potential of Jade-Seq™ for detecting rare mutations and determining the mutagenicity of environmental mutagens. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
209. Genome Engineering in Rice: Applications, Advancements and Future Perspectives
- Author
-
Tiwari, Shalini, Lata, Charu, Agarwal, Avinash Kumar, Series Editor, Singh, Sudhir P., editor, Upadhyay, Santosh Kumar, editor, Pandey, Ashutosh, editor, and Kumar, Sunil, editor
- Published
- 2019
- Full Text
- View/download PDF
210. Aptamers: Novel Therapeutic and Diagnostic Molecules
- Author
-
Singhal, Paavan, Gill, Anita Rani, Sharma, Preeti K., Kumar, Rakesh, Bhusal, Nabin, Kaur, Amandeep, Sharma, Pooja, Yadav, Gulab Singh, editor, Kumar, Vikas, editor, and Aggarwal, Neeraj K., editor
- Published
- 2019
- Full Text
- View/download PDF
211. Filaggrin-deficient rats generated using zinc-finger nucleases spontaneously exhibit dry scaly skin.
- Author
-
Nakashima, Chisa, Doi, Hiromi, Nakajima, Saeko, Mashimo, Tomoji, Oga, Toru, Ishida-Yamamoto, Akemi, Honda, Tetsuya, Ishida, Yoshihiro, Otsuka, Atsushi, and Kabashima, Kenji
- Subjects
- *
NUCLEASES , *RATS - Published
- 2022
- Full Text
- View/download PDF
212. The IRE1a-endonuclease regulates PD-1 expression through a novel XBP1/miRNA-34a axis within Natural Killer cells
- Subjects
Nucleases ,Killer cells ,Cancer ,Physical fitness ,Health - Abstract
2023 MAR 18 (NewsRx) -- By a News Reporter-Staff News Editor at Obesity, Fitness & Wellness Week -- According to news reporting based on a preprint abstract, our journalists obtained [...]
- Published
- 2023
213. mCas, a novel class of miniature type-V Cas12f nucleases with diverse PAM (Updated February 16, 2023)
- Subjects
Nucleases ,DNA binding proteins ,Physical fitness ,Health - Abstract
2023 MAR 11 (NewsRx) -- By a News Reporter-Staff News Editor at Obesity, Fitness & Wellness Week -- According to news reporting based on a preprint abstract, our journalists obtained [...]
- Published
- 2023
214. Casm, a novel class of miniature type-V Cas12f nucleases with diverse PAM
- Subjects
Nucleases ,DNA binding proteins ,Physical fitness ,Health - Abstract
2023 FEB 25 (NewsRx) -- By a News Reporter-Staff News Editor at Obesity, Fitness & Wellness Week -- According to news reporting based on a preprint abstract, our journalists obtained [...]
- Published
- 2023
215. New Enzymes and Coenzymes Study Findings Have Been Reported by a Researcher at Karolinska Institute (ATX-LPA-Dependent Nuclear Translocation of Endonuclease G in Respiratory Epithelial Cells: A New Mode Action for DNA Damage Induced by ...)
- Subjects
DNA ,Nucleases ,DNA damage ,Enzymes ,Physical fitness ,Health - Abstract
2023 FEB 18 (NewsRx) -- By a News Reporter-Staff News Editor at Obesity, Fitness & Wellness Week -- Researchers detail new data in enzymes and coenzymes. According to news originating [...]
- Published
- 2023
216. Activatable MRI probes for the specific detection of bacteria.
- Author
-
Periyathambi, Prabu, Balian, Alien, Hu, Zhangjun, Padro, Daniel, Hernandez, Luiza I., Uvdal, Kajsa, Duarte, Joao, and Hernandez, Frank J.
- Subjects
- *
MAGNETIC resonance imaging , *CONTRAST media , *FLUORESCENT probes , *MAGNETOTACTIC bacteria , *NUCLEIC acid probes - Abstract
Activatable fluorescent probes have been successfully used as molecular tools for biomedical research in the last decades. Fluorescent probes allow the detection of molecular events, providing an extraordinary platform for protein and cellular research. Nevertheless, most of the fluorescent probes reported are susceptible to interferences from endogenous fluorescence (background signal) and limited tissue penetration is expected. These drawbacks prevent the use of fluorescent tracers in the clinical setting. To overcome the limitation of fluorescent probes, we and others have developed activatable magnetic resonance probes. Herein, we report for the first time, an oligonucleotide-based probe with the capability to detect bacteria using magnetic resonance imaging (MRI). The activatable MRI probe consists of a specific oligonucleotide that targets micrococcal nuclease (MN), a nuclease derived from Staphylococcus aureus. The oligonucleotide is flanked by a superparamagnetic iron oxide nanoparticle (SPION) at one end, and by a dendron functionalized with several gadolinium complexes as enhancers, at the other end. Therefore, only upon recognition of the MRI probe by the specific bacteria is the probe activated and the MRI signal can be detected. This approach may be widely applied to detect bacterial infections or other human conditions with the potential to be translated into the clinic as an activatable contrast agent. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
217. RGEN-seq for highly sensitive amplification-free screen of off-target sites of gene editors.
- Author
-
Kuzin, Alexander, Redler, Brendan, Onuska, Jaya, and Slesarev, Alexei
- Subjects
- *
GENE therapy , *NUCLEOTIDE sequencing , *NUCLEASES , *POLLUTANTS , *GENES - Abstract
Sensitive detection of off-target sites produced by gene editing nucleases is crucial for developing reliable gene therapy platforms. Although several biochemical assays for the characterization of nuclease off-target effects have been recently published, significant technical and methodological issues still remain. Of note, existing methods rely on PCR amplification, tagging, and affinity purification which can introduce bias, contaminants, sample loss through handling, etc. Here we describe a sensitive, PCR-free next-generation sequencing method (RGEN-seq) for unbiased detection of double-stranded breaks generated by RNA-guided CRISPR-Cas9 endonuclease. Through use of novel sequencing adapters, the RGEN-Seq method saves time, simplifies workflow, and removes genomic coverage bias and gaps associated with PCR and/or other enrichment procedures. RGEN-seq is fully compatible with existing off-target detection software; moreover, the unbiased nature of RGEN-seq offers a robust foundation for relating assigned DNA cleavage scores to propensity for off-target mutations in cells. A detailed comparison of RGEN-seq with other off-target detection methods is provided using a previously characterized set of guide RNAs. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
218. Expanding the range of editable targets in the wheat genome using the variants of the Cas12a and Cas9 nucleases.
- Author
-
Wang, Wei, Tian, Bin, Pan, Qianli, Chen, Yueying, He, Fei, Bai, Guihua, Akhunova, Alina, Trick, Harold N., and Akhunov, Eduard
- Subjects
- *
RNA polymerase II , *SWITCHGRASS , *NUCLEASES , *GENOMES , *TRANSGENIC plants , *LOCUS (Genetics) - Abstract
Summary: The development of CRISPR‐based editors recognizing distinct protospacer‐adjacent motifs (PAMs), or having different spacer length/structure requirements broadens the range of possible genomic applications. We evaluated the natural and engineered variants of Cas12a (FnCas12a and LbCas12a) and Cas9 for their ability to induce mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a‐induced mutations in the wheat genome, even though with a lower rate than that reported for SpCas9. The eight‐fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guides flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides and showed substantial increase after subjecting transgenic plants to high‐temperature treatment. We successfully applied LbCas12a‐MGE for generating heritable mutations in a gene controlling grain size and weight in wheat. We showed that the range of editable loci in the wheat genome could be further expanded by using the engineered variants of Cas12a (LbCas12a‐RVR) and Cas9 (Cas9‐NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9‐NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports a set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to modification using the original SpCas9 nuclease. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
219. Helical filament structure of the DREP3 CIDE domain reveals a unified mechanism of CIDE‐domain assembly.
- Author
-
Lee, So Yeon, Kwon, Sunghark, Ha, Hyun Ji, Lee, Sung Hoon, and Park, Hyun Ho
- Subjects
- *
HELICAL structure , *MOLECULAR size , *LIPID metabolism , *HOMEOSTASIS , *CELL death , *NUCLEASES - Abstract
The cell‐death‐inducing DFF45‐like effector (CIDE) domain is a protein‐interaction module comprising ∼80 amino acids and was initially identified in several apoptotic nucleases and their regulators. CIDE‐domain‐containing proteins were subsequently identified among proteins involved in lipid metabolism. Given the involvement of CIDE‐domain‐containing proteins in cell death and lipid homeostasis, their structure and function have been intensively studied. Here, the head‐to‐tail helical filament structure of the CIDE domain of DNA fragmentation factor‐related protein 3 (DREP3) is presented. The helical filament structure was formed by opposing positively and negatively charged interfaces of the domain and was assembled depending on protein and salt concentrations. Although conserved filament structures are observed in CIDE family members, the structure elucidated in this study and its comparison with previous structures indicated that the size and the number of molecules used in one turn vary. These findings suggest that this charged‐surface‐based head‐to‐tail helical filament structure represents a unified mechanism of CIDE‐domain assembly and provides insight into the function of various forms of the filament structure of the CIDE domain in higher‐order assembly for apoptotic DNA fragmentation and control of lipid‐droplet size. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
220. Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells.
- Author
-
Bigelyte, Greta, Young, Joshua K., Karvelis, Tautvydas, Budre, Karolina, Zedaveinyte, Rimante, Djukanovic, Vesna, Van Ginkel, Elizabeth, Paulraj, Sushmitha, Gasior, Stephen, Jones, Spencer, Feigenbutz, Lanie, Clair, Grace St., Barone, Pierluigi, Bohn, Jennifer, Acharya, Ananta, Zastrow-Hayes, Gina, Henkel-Heinecke, Selgar, Silanskas, Arunas, Seidel, Ralf, and Siksnys, Virginijus
- Subjects
NUCLEASES ,DNA ,HEAT pulses ,GENOME editing ,EUKARYOTIC cells ,CRISPRS - Abstract
Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools. Miniature Cas12f editing systems are well suited for in vivo editing applications. Here the authors characterize the intrinsic activity of SpCas12f1 in plant and animal cells. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
221. PRIMA: a rapid and cost-effective genotyping method to detect single-nucleotide differences using probe-induced heteroduplexes.
- Author
-
Kakui, Hiroyuki, Yamazaki, Misako, and Shimizu, Kentaro K.
- Subjects
- *
SINGLE-stranded DNA , *SITE-specific mutagenesis , *DNA probes , *DNA , *NUCLEASES - Abstract
Targeted mutagenesis by programmable site-specific nucleases like CRISPR typically produce 1-base pair (bp) insertion or deletion (indel) mutations. Although several methods have been developed to detect such 1-bp indels, each method has pros and cons in terms of cost and/or resolution. Heteroduplex mobility assay (HMA) is a traditional technique detecting small base pair differences but it has a limited resolution of mutation size and the band patterns are often complex. Here, we developed a new method called PRIMA (Probe-Induced HMA) using a short single-stranded DNA molecule as a probe in HMA. By utilizing a 40-mer probe containing a 5-nucleotide deletion, we assessed the mobility of a heteroduplex with a target DNA fragment from a plant, bacterium, and human. This method allowed us to detect a 1-bp indel mutation consistently. We also showed that SNPs can be detected using PRIMA. PRIMA provides a rapid and cost-effective solution for the genotyping. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
222. Stage-Specific Class I Nucleases of Leishmania Play Important Roles in Parasite Infection and Survival.
- Author
-
Freitas-Mesquita, Anita Leocadio and Meyer-Fernandes, José Roberto
- Subjects
LEISHMANIASIS ,NUCLEASES ,NEGLECTED diseases ,LEISHMANIA ,ALIMENTARY canal ,PARASITES ,ADENOSINES - Abstract
Protozoans of the genus Leishmania are the causative agents of an important neglected tropical disease referred to as leishmaniasis. During their lifecycle, the parasites can colonize the alimentary tract of the sand fly vector and the parasitophorous vacuole of the mammalian host, differentiating into distinct stages. Motile promastigotes are found in the sand fly vector and are transmitted to the mammalian host during the insect blood meal. Once in the vertebrate host, the parasites differentiate into amastigotes and multiply inside macrophages. To successfully establish infection in mammalian hosts, Leishmania parasites exhibit various strategies to impair the microbicidal power of the host immune system. In this context, stage-specific class I nucleases play different and important roles related to parasite growth, survival and development. Promastigotes express 3'-nucleotidase/nuclease (3'-NT/NU), an ectoenzyme that can promote parasite escape from neutrophil extracellular traps (NET)-mediated death through extracellular DNA hydrolysis and increase Leishmania -macrophage interactions due to extracellular adenosine generation. Amastigotes express secreted nuclease activity during the course of human infection that may be involved in the purine salvage pathway and can mobilize extracellular nucleic acids available far from the parasite. Another nuclease expressed in amastigotes (P4/LmC1N) is located in the endoplasmic reticulum of the parasite and may be involved in mRNA stability and DNA repair. Homologs of this class I nuclease can induce protection against infection by eliciting a T helper 1-like immune response. These immunogenic properties render these nucleases good targets for the development of vaccines against leishmaniasis, mainly because amastigotes are the form responsible for the development and progression of the disease. The present review aims to present and discuss the roles played by different class I nucleases during the Leishmania lifecycle, especially regarding the establishment of mammalian host infection. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
223. In vitro analysis of site specific nuclease selectivity by NGS.
- Author
-
Brondani, Vincent
- Subjects
- *
MOLECULAR interactions , *ENDONUCLEASES , *NUCLEOTIDE sequence , *NUCLEASES , *DNA sequencing , *DNA - Abstract
Nucleases currently used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. The RNA guided nucleases describe today are recognizing a sequence with two distinct molecular interactions: first, like a restriction endonuclease, by direct interaction between the protein and the DNA; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report an in vitro assay to assess the cleavage specificity and the selectivity of the nucleases. The assay is designed using a plasmid encompassing the DNA target site degenerated at positions determined on structural feature. The results demonstrate that the Cpf1 RNA guided nuclease is highly specific for the target sequence, nevertheless its substrate selectivity is low compare to a restriction endonuclease. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
224. Harnessing the power of directed evolution to improve genome editing systems.
- Author
-
Su, Qiwen, Zhou, Mi, Cheng, Cristina, and Niu, Jia
- Subjects
- *
GENOME editing , *NUCLEASES , *GENOMES - Abstract
The recent development of genome editing systems, such as zinc-finger nucleases, transcription activator–like effectors, CRISPR-Cas nucleases, and base editors has enabled the unprecedented capability to engineer the genomes of living cells. The ever-increasing demand for genome editors with improved accuracy, activity, and functionality has stimulated significant efforts to further engineer the genome editing systems. Directed evolution represents a promising strategy to improve the existing genome editing systems and enable new editing functions. Here, we review recent representative strategies to harness the power of directed evolution to improve genome editing systems, which have led to state-of-the-art genome editors that have significant implications for diverse applications in both laboratories and clinics. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
225. The core Cas1 protein of CRISPR-Cas I-B in Leptospira shows metal-tunable nuclease activity
- Author
-
Bhuvan Dixit, Aman Prakash, Pankaj Kumar, Prerana Gogoi, and Manish Kumar
- Subjects
Leptospira ,Nucleases ,Cas protein ,CRISPR-Cas ,DNase ,Microbiology ,QR1-502 ,Genetics ,QH426-470 - Abstract
Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 is the causative agent of leptospirosis in animals and humans. This organism carries a functional cas1 gene classified under CRISPR-Cas I-B. In this study, using various nuclease assays and bioinformatics analysis, we report that the recombinant Cas1 (LinCas1) possesses metal-ion dependent DNase activity, which is inhibited upon substitution or chelation of metal-ion and/or interaction with recombinant Cas2 (LinCas2) of L. interrogans. Model of LinCas1 structure shows a shorter N-terminal domain unlike other Cas1 orthologs reported to date. The C-terminal domain of LinCas1 contains conserved divalent-metal binding residues (Glu108, His176, and Glu191) and the mutation of these residues leads to abolition in DNase activity. Immunoassay using anti-LinCas2 demonstrates that LinCas1 interacts with LinCas2 and attains a saturation point. Moreover, the nuclease activity of the LinCas1-Cas2 mixture on ds-DNA displayed a reduction in activity compared to the pure core LinCas proteins under in vitro condition. The DNase activity for LinCas1 is consistent with a role for this protein in the recognition/cleavage of foreign DNA and integration of foreign DNA as spacer into the CRISPR array.
- Published
- 2021
- Full Text
- View/download PDF
226. CoCoNuTs are a diverse subclass of Type IV restriction systems predicted to target RNA.
- Author
-
Bell RT, Sahakyan H, Makarova KS, Wolf YI, and Koonin EV
- Subjects
- Phylogeny, Bacterial Proteins metabolism, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacteria genetics, Bacteria metabolism, RNA metabolism, RNA genetics, RNA chemistry, RNA, Bacterial metabolism, RNA, Bacterial chemistry, RNA, Bacterial genetics
- Abstract
A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote co iled- co il nu clease t andems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF ( C RISPR- A ssociated R ossmann F old) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over., Competing Interests: RB, HS, KM, YW, EK No competing interests declared
- Published
- 2024
- Full Text
- View/download PDF
227. Patent Issued for Optimized CRISPR-Cas nucleases and base editors and methods of use thereof (USPTO 11913005)
- Subjects
Editors ,Nucleases ,DNA binding proteins ,Biotechnology industry ,Pharmaceuticals and cosmetics industries - Abstract
2024 MAR 20 (NewsRx) -- By a News Reporter-Staff News Editor at Biotech Week -- According to news reporting originating from Alexandria, Virginia, by NewsRx journalists, a patent by the [...]
- Published
- 2024
228. Patent Issued for Treatment of diseases by liver expression of an enzyme which has a deoxyribonuclease (DNase) activity (USPTO 11905522)
- Subjects
Nucleases ,Liver ,Enzymes -- Intellectual property ,Liver diseases ,Business ,Health ,Health care industry - Abstract
2024 MAR 12 (NewsRx) -- By a News Reporter-Staff News Editor at Cancer Weekly -- CLS Therapeutics Limited (Saint Peter Port, Guernsey) has been issued patent number 11905522, according to [...]
- Published
- 2024
229. Patent Issued for Nucleic acid sequence replacement by NHEJ (USPTO 11891635)
- Subjects
Nucleases ,RNA ,DNA binding proteins ,Biotechnology industry ,Pharmaceuticals and cosmetics industries - Abstract
2024 FEB 28 (NewsRx) -- By a News Reporter-Staff News Editor at Biotech Week -- Max-Delbruck-Centrum fur Molekulare Medizin in der Helmholtz-Gemeinschaft (Berlin, Germany) has been issued patent number 11891635, [...]
- Published
- 2024
230. A multi-transcriptomics approach reveals the coordinated action of the endoribonuclease DNE1 and the decapping machinery in orchestrating mRNA decay
- Subjects
Machinery ,Arabidopsis thaliana ,Messenger RNA ,Nucleases ,Enzymes ,Magneto-electric machines ,Biological sciences ,Health - Abstract
2024 FEB 20 (NewsRx) -- By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting based on a preprint abstract, our journalists obtained the following [...]
- Published
- 2024
231. Integrated Biobank of Luxembourg Researchers Focus on Science and Technology (Implementing routine monitoring for nuclease contamination of equipment and consumables into the quality Management system of a laboratory)
- Subjects
Nucleases ,Quality control ,Quality control ,Health ,Science and technology - Abstract
2024 FEB 9 (NewsRx) -- By a News Reporter-Staff News Editor at Science Letter -- New research on science and technology is the subject of a new report. According to [...]
- Published
- 2024
232. Researchers Submit Patent Application, 'Polycyclic Cap-Dependent Endonuclease Inhibitors For Treating Or Preventing Influenza', for Approval (USPTO 20240018145)
- Subjects
Nucleases ,Influenza -- Prevention ,Vaccines -- Intellectual property ,Health - Abstract
2024 FEB 7 (NewsRx) -- By a News Reporter-Staff News Editor at Vaccine Weekly -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors Bennett, Frank [...]
- Published
- 2024
233. Flap endonuclease Substrate Interactions studied using Dynamic Single-Molecule Atomic Force Microscopy (Updated January 17, 2024)
- Subjects
Microscope and microscopy ,Nucleases ,Atomic force microscopy ,Biological sciences ,Health - Abstract
2024 JAN 30 (NewsRx) -- By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting based on a preprint abstract, our journalists obtained the following [...]
- Published
- 2024
234. 'Optimized Engineered Nucleases Having Specificity For The Human T Cell Receptor Alpha Constant Region Gene' in Patent Application Approval Process (USPTO 20240000845)
- Subjects
T cells ,Nucleases ,Amino acids -- Intellectual property ,Genetic engineering ,DNA binding proteins ,Business ,Health - Abstract
2024 JAN 26 (NewsRx) -- By a News Reporter-Staff News Editor at Medical Patent Business Week -- A patent application by the inventors Genschel, Jochen (Raleigh, NC, US); Lape, Janel [...]
- Published
- 2024
235. 'Inducible, Tunable, and Multiplex Human Gene Regulation Using CRISPR-Cpf1' in Patent Application Approval Process (USPTO 20230416310)
- Subjects
Nucleases ,DNA binding proteins ,Health - Abstract
2024 JAN 19 (NewsRx) -- By a News Reporter-Staff News Editor at Health & Medicine Week -- A patent application by the inventors Joung, J. Keith (Winchester, MA, US); Kleinstiver, [...]
- Published
- 2024
236. Non-CRISPR Genome Editing Therapy Market projected to reach USD 1,228.0 Million by 2030, growing at a CAGR of 16.3% during the forecast period of 2023-2030 - pronounced by MarketDigits in its recent study
- Subjects
Zinc finger proteins ,Genomics ,Nervous system diseases ,Genomes ,Nucleases ,Genetic transcription ,DNA binding proteins ,General interest ,News, opinion and commentary - Abstract
Richmond: PIONEERING MARKETDIGITS CONSULTING AND ADVISORY PRIVATE LIMITED has issued the following news release: According to a research report 'Non-CRISPR Genome Editing Therapy Market', by Techniques (Zinc Finger Nucleases (ZFNs), [...]
- Published
- 2023
237. Profile nucleases and Repurpose Off-Targets to Expand Gene Editing
- Subjects
Genomics ,Genes ,Nucleases ,DNA binding proteins ,Business, international ,European Union - Abstract
Profile Nucleases And Repurpose Off-Targets To Expand Gene Editing Objective Programmable nucleases reside at the nexus of advanced gene editing and microbial defense. These nucleases degrade phage DNA but avoid [...]
- Published
- 2023
238. Unique Clindamycin-Resistant Clostridioides difficile Strain Related to Fluoroquinolone-Resistant Epidemic BI/RT027 Strain
- Author
-
Skinner, Andrew M., Petrella, Laurica, Siddiqui, Farida, Sambol, Susan P., Gulvik, Christopher A., Gerding, Dale N., Donskey, Curtis J., and Johnson, Stuart
- Subjects
Hospital patients -- Analysis ,Epidemics -- Illinois -- Ohio -- Analysis ,Long term care facilities -- Analysis ,Clindamycin -- Analysis ,DNA sequencing -- Analysis ,Medical research -- Analysis ,Long term care -- Analysis ,Genomes -- Analysis ,Genomics -- Analysis ,Nucleases ,Intelligence gathering ,Antibacterial agents ,Moxifloxacin ,Diseases ,Genes ,Health - Abstract
As the leading cause of healthcare-associated infectious diarrhea and colitis, Clostridioides (formerly Clostridium) difficile continues to affect patients in hospitals and extended-care facilities in the United States (1-3). Among the [...]
- Published
- 2020
- Full Text
- View/download PDF
239. Human-to-Human Transmission of Influenza A(H3N2) Virus with Reduced Susceptibility to Baloxavir, Japan, February 2019
- Author
-
Takashita, Emi, Ichikawa, Masataka, Morita, Hiroko, Ogawa, Rie, Fujisaki, Seiichiro, Shirakura, Masayuki, Miura, Hideka, Nakamura, Kazuya, Kishida, Noriko, Kuwahara, Tomoko, Sugawara, Hiromi, Sato, Aya, Akimoto, Miki, Mitamura, Keiko, Abe, Takashi, Yamazaki, Masahiko, Watanabe, Shinji, Hasegawa, Hideki, and Odagiri, Takato
- Subjects
Influenza viruses ,Laninamivir ,Disease susceptibility ,Influenza ,Peramivir ,Genomics ,Genes ,Genomes ,Nucleases ,Infection ,Clinical trials ,Baloxavir marboxil ,Health - Abstract
The cap-dependent endonuclease inhibitor baloxavir marboxil is approved in Japan for the treatment of influenza virus infection in patients >12 years of age and children In phase 3 trials, patients [...]
- Published
- 2019
- Full Text
- View/download PDF
240. Synthesis and Properties of Oligonucleotides Containing 2′‐O,4′‐C‐Ethylene‐Bridged 5‐Methyluridine with Exocyclic Methylene and Methyl Groups in the Bridge.
- Author
-
Ito, Yuta, Nishida, Kodai, Tsutsui, Norika, Fuchi, Yasufumi, and Hari, Yoshiyuki
- Subjects
- *
OLIGONUCLEOTIDE synthesis , *METHYL groups , *NUCLEIC acids , *OLIGONUCLEOTIDES , *METHYLENE group , *NUCLEASES - Abstract
Novel 2′‐O,4′‐C‐ethylene‐bridged nucleic acid (ENA) analogs with exocyclic methylene and (R)‐methyl groups at the 6′‐position were synthesized. The obtained 6′‐methylene‐ENA and 6′R‐Me‐ENA were incorporated into oligonucleotides, and their properties were compared with those of the corresponding natural, ENA‐, and 6′S‐Me‐ENA‐modified oligonucleotides. The duplex‐stabilizing ability of 6′‐methylene‐ENA and 6′R‐Me‐ENA with single‐stranded RNA (ssRNA) was similar to that of ENA and 6′S‐Me‐ENA under both dilute and molecular crowding conditions. On the other hand, oligonucleotides containing 6′‐methylene‐ENA and 6′R‐Me‐ENA formed stable triplexes with double‐stranded DNA (dsDNA) in comparison with those containing 6′S‐Me‐ENA. Their thermodynamic properties were also calculated to explore the influence of the substituents introduced at the 6′‐position of ENA. Moreover, oligonucleotides with ENA analogs exhibited excellent resistance against nucleases. These results suggest that 6′‐methylene‐ENA and 6′R‐Me‐ENA are promising candidates for targeting ssRNA and dsDNA. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
241. Direct sequencing of measles virus complete genomes in the midst of a large-scale outbreak.
- Author
-
Bucris, Efrat, Indenbaum, Victoria, Azar, Roberto, Erster, Oran, Haas, Eric, Mendelson, Ella, and Zuckerman, Neta S.
- Subjects
- *
VIRAL genomes , *MEASLES virus , *NUCLEOTIDE sequencing , *NUCLEASES , *GENOMES , *GENOTYPES - Abstract
Measles outbreaks escalated globally despite worldwide elimination efforts. Molecular epidemiological investigations utilizing partial measles virus (MeV) genomes are challenged by reduction in global genotypes and low evolutionary rates. Greater resolution was reached using MeV complete genomes, however time and costs limit the application to numerous samples. We developed an approach to unbiasedly sequence complete MeV genomes directly from patient urine samples. Samples were enriched for MeV using filtration or nucleases and the minimal number of sequence reads to allocate per sample based on its MeV content was assessed using in-silico reduction of sequencing depth. Application of limited-resource sequencing to treated MeV-positive samples demonstrated that 1–5 million sequences for samples with high/medium MeV quantities and 10–15 million sequences for samples with lower MeV quantities are sufficient to obtain >98% MeV genome coverage and over X50 average depth. This approach enables real-time high-resolution molecular epidemiological investigations of large-scale MeV outbreaks. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
242. FANCD2-Associated Nuclease 1 Partially Compensates for the Lack of Exonuclease 1 in Mismatch Repair.
- Author
-
Kratz, Katja, Artola-Borán, Mariela, Kobayashi-Era, Saho, Koh, Gene, Oliveira, Goncalo, Kobayashi, Shunsuke, Oliveira, Andreia, Xueqing Zou, Richter, Julia, Masataka Tsuda, Hiroyuki Sasanuma, Shunichi Takeda, Loizou, Joanna I., Sartori, Alessandro A., Nik-Zainal, Serena, and Jiricny, Josef
- Subjects
- *
EUKARYOTIC cells , *PHENOTYPES , *NUCLEASES , *DRUG resistance , *COLON cancer - Abstract
Germline mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1, and PMS2 are linked to cancer of the colon and other organs, characterized by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in EXO1, encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1. Analysis of the MLH1 interactome identified FANCD2-associated nuclease 1 (FAN1), a novel enzyme with biochemical properties resembling EXO1. We now show that FAN1 efficiently substitutes for EXO1 in MMR assays and that this functional complementation is modulated by its interaction with MLH1. FAN1 also contributes to MMR in vivo; cells lacking both EXO1 and FAN1 have an MMR defect and display resistance to Nmethyl-N-nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, suggesting that the two nucleases act redundantly in the same antimutagenic pathway. However, the increased drug resistance and mutator phenotype of FAN1/EXO1-deficient cells are less prominent than those seen in cells lacking MSH6 or MLH1. Eukaryotic cells thus apparently possess additional mechanisms that compensate for the loss of EXO1. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
243. RNAi Technology in Fish and Shellfish-Status and Prospects: A Review.
- Author
-
Qayoom, Ubaid and Mushtaq, Zahoor
- Subjects
- *
SMALL interfering RNA , *FISH farming , *RNA , *AQUATIC animals , *NUCLEASES - Abstract
Ribonucleic acid interference (RNAi), a valuable tool for manipulating gene functionality in the laboratory, has also emerged as a powerful tool to suppress infection or replication of many pathogens that cause severe economic losses in fish farming. By taking advantage of the cell's endogenous RNAi apparatus, small interfering RNA of ~21-22 bp can be introduced into cells to induce target specific mRNA degradation. With the growing appreciation for the potential of RNAi technology, the diversity in vivo relevance to aquaculture is seemingly vast. Studies in the future should address the hurdles like delivery strategy stability and degradation of RNAi therapeutic molecule by nucleases in aquatic animals. In this article, we review the literature in the field of RNAi technology in aquaculture, summarize the status and prospects, which may open doors to its applicability potential as a therapeutic strategy to modulate host-pathogen interactions and inspire further trials. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
244. TALENs—an indispensable tool in the era of CRISPR: a mini review.
- Author
-
Bhardwaj, Anuradha and Nain, Vikrant
- Subjects
CRISPRS ,DNA restriction enzymes ,GENOME editing ,MITOCHONDRIAL DNA ,ENDONUCLEASES ,SYNTHETIC biology ,NUCLEASES - Abstract
Background: Genome of an organism has always fascinated life scientists. With the discovery of restriction endonucleases, scientists were able to make targeted manipulations (knockouts) in any gene sequence of any organism, by the technique popularly known as genome engineering. Though there is a range of genome editing tools, but this era of genome editing is dominated by the CRISPR/Cas9 tool due to its ease of design and handling. But, when it comes to clinical applications, CRISPR is not usually preferred. In this review, we will elaborate on the structural and functional role of designer nucleases with emphasis on TALENs and CRISPR/Cas9 genome editing system. We will also present the unique features of TALENs and limitations of CRISPRs which makes TALENs a better genome editing tool than CRISPRs. Main body: Genome editing is a robust technology used to make target specific DNA modifications in the genome of any organism. With the discovery of robust programmable endonucleases-based designer gene manipulating tools such as meganucleases (MN), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein (CRISPR/Cas9), the research in this field has experienced a tremendous acceleration giving rise to a modern era of genome editing with better precision and specificity. Though, CRISPR-Cas9 platform has successfully gained more attention in the scientific world, TALENs and ZFNs are unique in their own ways. Apart from high-specificity, TALENs are proven to target the mitochondrial DNA (mito-TALEN), where gRNA of CRISPR is difficult to import. This review talks about genome editing goals fulfilled by TALENs and drawbacks of CRISPRs. Conclusions: This review provides significant insights into the pros and cons of the two most popular genome editing tools TALENs and CRISPRs. This mini review suggests that, TALENs provides novel opportunities in the field of therapeutics being highly specific and sensitive toward DNA modifications. In this article, we will briefly explore the special features of TALENs that makes this tool indispensable in the field of synthetic biology. This mini review provides great perspective in providing true guidance to the researchers working in the field of trait improvement via genome editing. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
245. Preparation of siRNA–PLGA/Fabʹ–PLGA mixed micellar system with target cell-specific recognition.
- Author
-
Hazekawa, Mai, Nishinakagawa, Takuya, Mori, Takeshi, Yoshida, Miyako, Uchida, Takahiro, and Ishibashi, Daisuke
- Subjects
- *
SMALL interfering RNA , *CELL membranes , *GENE delivery techniques , *GLYCOLIC acid , *NUCLEASES - Abstract
Small interfering RNAs (siRNAs) are susceptible to nucleases and degrade quickly in vivo. Moreover, siRNAs demonstrate poor cellular uptake and cannot cross the cell membrane because of its polyanionic characteristics. To overcome these challenges, an intelligent gene delivery system that protects siRNAs from nucleases and facilitates siRNA cellular uptake is required. We previously reported the potential of siRNA-poly(d,l-lactic-co-glycolic acid; PLGA) micelles as an effective siRNA delivery tool in a murine peritoneal dissemination model by local injection. However, there was no effective formulation for siRNA delivery to target cells via intravenous injection. This study aimed to prepare siRNA–PLGA/Fabʹ–PLGA mixed micelles for siRNA delivery to target floating cells and evaluate its formulation in vitro. As the target siRNA protein in CEMx174, CyclinB1 levels were significantly reduced when siRNA–PLGA/Fabʹ–PLGA mixed micelles were added to cells compared with siRNA–PLGA micelles. siRNA–PLGA/Fabʹ–PLGA mixed micelles have high cell permeability and high target cell accumulation by endocytosis because flow cytometry detected labeling micelles in target cells. This study supports siRNA–PLGA/Fabʹ–PLGA mixed micelles as an effective siRNA delivery tool. This formulation can be administered systemically in dosage form against target cells, including cancer metastasis or blood cancer. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
246. Development of CRISPR_Cas9 genome editing system and its application in rice molecular breeding.
- Author
-
Ma, Zengfeng, Wei, Minyi, Zhang, Yuexiong, Qin, Gang, Liu, Chi, Li, Zhenjing, and Huang, Dahui
- Subjects
- *
GENOME editing , *CRISPRS , *RICE breeding , *GENETIC engineering , *DNA sequencing , *NUCLEIC acids , *NUCLEASES - Abstract
Gene editing is a new genetic engineering technology that uses sequence-specific nucleases to insert, delete, or replace nucleic acid base(s) of the target gene, or introduce exogenous DNA sequences at a specific site in the genome of an organism to achieve the desired modification. Currently, the CRISPR/Cas system is the most widely used genome editing technology because it is relatively simple with high editing efficiency. In this review, we discuss the CRISPR/Cas system's application and its current limitations in rice breeding. Also, we elaborate on possible improved methods regarding its mechanism and development history. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
247. CRISPR-Cas9 and beyond: what's next in plant genome engineering.
- Author
-
Zess, Erin and Begemann, Matthew
- Subjects
- *
PLANT genomes , *CRISPRS , *GENOME editing , *ZINC-finger proteins , *NUCLEASES , *PLANT spacing - Abstract
Scientists have developed and deployed successive generations of genome engineering technologies for use in plants, including meganucleases, zinc finger nucleases, TAL effector nucleases, and CRISPR nucleases. Each of these tools has been hailed as potentially revolutionary, capable of providing more efficient and precise ways to modify plant genomes toward improving agronomic traits or making fundamental discoveries. The CRISPR nucleases, in particular, have accelerated the pace of innovation and expanded the boundaries of what is achievable within the plant research space. This review will take care to discuss current plant genome engineering technologies, covering both well-established and up-and-coming tools, as well as describe potential and real-world applications. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
248. Genome Editing Technology and Its Application Potentials in the Industrial Filamentous Fungus Aspergillus oryzae.
- Author
-
Jun-ichi Maruyama
- Subjects
- *
GENOME editing , *KOJI , *FILAMENTOUS fungi , *BIOSYNTHESIS , *CRISPRS , *NUCLEASES - Abstract
Aspergillus oryzae is a filamentous fungus that has been used in traditional Japanese brewing industries, such as the sake, soy sauce, and miso production. In addition, A. oryzae has been used in heterologous protein production, and the fungus has been recently used in biosynthetic research due to its ability to produce a large amount of heterologous natural products by introducing foreign biosynthetic genes. Genetic manipulation, which is important in the functional development of A. oryzae, has mostly been limited to the wild strain RIB40, a genome reference suitable for laboratory analysis. However, there are numerous industrial brewing strains of A. oryzae with various specialized characteristics, and they are used selectively according to the properties required for various purposes such as sake, soy sauce, and miso production. Since the early 2000s, genome editing technologies have been developed; among these technologies, transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPRassociated protein 9 (CRISPR/Cas9) have been applied to gene modification in A. oryzae. Notably, the CRISPR/Cas9 system has dramatically improved the efficiency of gene modification in industrial strains of A. oryzae. In this review, the development of genome editing technology and its application potentials in A. oryzae are summarized. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
249. Production of Transgenic Handmade Cloned Goat (Capra hircus) Embryos by Targeted Integration into Rosa 26 Locus Using Transcription Activator-like Effector Nucleases.
- Author
-
Vats, Preeti, Kaushik, Ramakant, Rawat, Nidhi, Sharma, Ankur, Sharma, Tushar, Dua, Diksha, Singh, Manoj Kumar, Palta, Prabhat, Singla, Suresh Kumar, Manik, Radhey Sham, and Chauhan, Manmohan Singh
- Subjects
- *
GOATS , *NUCLEASES , *EMBRYOS , *PHENOTYPES , *SOMATIC cell nuclear transfer , *MAMMARY glands , *GENE targeting - Abstract
Transgenic goats are ideal bioreactors for the production of therapeutic proteins in their mammary glands. However, random integration of the transgene within-host genome often culminates in unstable expression and unpredictable phenotypes. Targeting desired genes to a safe locus in the goat genome using advanced targeted genome-editing tools, such as transcription activator-like effector nucleases (TALENs) might assist in overcoming these hurdles. We identified Rosa 26 locus, a safe harbor for transgene integration, on chromosome 22 in the goat genome for the first time. We further demonstrate that TALEN-mediated targeting of GFP gene cassette at Rosa 26 locus exhibited stable and ubiquitous expression of GFP gene in goat fetal fibroblasts (GFFs) and after that, transgenic cloned embryos generated by handmade cloning (HMC). The transfection of GFFs by the TALEN pair resulted in 13.30% indel frequency at the target site. Upon cotransfection with TALEN and donor vectors, four correctly targeted cell colonies were obtained and all of them showed monoallelic gene insertions. The blastocyst rate for transgenic cloned embryos (3.92% ± 1.12%) was significantly (p < 0.05) lower than cloned embryos (7.84% ± 0.68%) used as control. Concomitantly, 2 out of 15 embryos of morulae and blastocyst stage (13.30%) exhibited site-specific integration. In conclusion, the present study demonstrates TALEN-mediated transgene integration at Rosa 26 locus in caprine fetal fibroblasts and the generation of transgenic cloned embryos using HMC. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
250. Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris.
- Author
-
Verhülsdonk, Lukas, Mannherz, Hans Georg, and Napirei, Markus
- Subjects
- *
CELL differentiation , *SKELETAL muscle , *NUCLEASES , *CELL death , *PICHIA pastoris , *HEPARIN - Abstract
Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris. Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.