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https://www.readbyqxmd.com/read/28819113/insights-into-the-role-of-endonuclease-v-in-rna-metabolism-in-trypanosoma-brucei
#1
Daniel García-Caballero, Guiomar Pérez-Moreno, Antonio M Estévez, Luis Miguel Ruíz-Pérez, Antonio E Vidal, Dolores González-Pacanowska
Inosine may arise in DNA as a result of oxidative deamination of adenine or misincorporation of deoxyinosine triphosphate during replication. On the other hand, the occurrence of inosine in RNA is considered a normal and essential modification induced by specific adenosine deaminases acting on mRNA and tRNA. In prokaryotes, endonuclease V (EndoV) can recognize and cleave inosine-containing DNA. In contrast, mammalian EndoVs preferentially cleave inosine-containing RNA, suggesting a role in RNA metabolism for the eukaryotic members of this protein family...
August 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28815907/t7-rna-polymerase-driven-inducible-cell-lysis-for-dna-transfer-from-escherichia-coli-to-bacillus-subtilis
#2
Mario Juhas, James W Ajioka
The majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis are therefore of the highest importance. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr-ts-1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E...
August 16, 2017: Microbial Biotechnology
https://www.readbyqxmd.com/read/28815851/basics-of-genome-editing-technology-and-its-application-in-livestock-species
#3
REVIEW
Bjoern Petersen
In the last decade, the research community has witnessed a blooming of targeted genome editing tools and applications. Novel programmable DNA nucleases such as zinc finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs) and the clustered regularly interspaced short palindromic repeats/Cas9 system (CRISPR/Cas9) possess long recognition sites and are capable of cutting DNA in a very specific manner. These DNA nucleases mediate targeted genetic alterations by enhancing the DNA mutation rate via induction of double-strand breaks at a predetermined genomic site...
August 2017: Reproduction in Domestic Animals, Zuchthygiene
https://www.readbyqxmd.com/read/28815504/purification-and-in-vitro-characterization-of-zinc-finger-recombinases
#4
Femi J Olorunniji, Susan J Rosser, W Marshall Stark
Zinc finger recombinases (ZFRs) are designer site-specific recombinases that have been adapted for a variety of genome editing purposes. Due to their modular nature, ZFRs can be customized for targeted sequence recognition and recombination. There has been substantial research on the in vivo properties and applications of ZFRs; however, in order to fully understand and customize them, it will be necessary to study their properties in vitro. Experiments in vitro can allow us to optimize catalytic activities, improve target specificity, measure and minimize off-target activity, and characterize key steps in the recombination pathway that might be modified to improve performance...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28815500/nanoparticle-mediated-recombinase-delivery-into-maize
#5
Susana Martin-Ortigosa, Brian G Trewyn, Kan Wang
We describe a non-DNA-based system for delivering Cre recombinase protein into maize tissue using gold-plated mesoporous silica nanoparticle (Au-MSN). Cre protein is first loaded into the pores of Au-MSNs and then delivered using the biolistic method to immature embryos of a maize line (Lox-corn), which harbors loxP sites flanking a selection and a reporter gene. The release of the Cre recombinase protein inside the plant cell leads to recombination at the loxP sites, eliminating both genes. Visual screening is used to identify recombination events, which can be regenerated to mature and fertile plants...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28814300/increasing-on-target-cleavage-efficiency-for-crispr-cas9-induced-large-fragment-deletion-in-myxococcus-xanthus
#6
Ying-Jie Yang, Ye Wang, Zhi-Feng Li, Ya Gong, Peng Zhang, Wen-Chao Hu, Duo-Hong Sheng, Yue-Zhong Li
BACKGROUND: The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. RESULTS: In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells...
August 16, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28813413/polylox-barcoding-reveals-haematopoietic-stem-cell-fates-realized-in-vivo
#7
Weike Pei, Thorsten B Feyerabend, Jens Rössler, Xi Wang, Daniel Postrach, Katrin Busch, Immanuel Rode, Kay Klapproth, Nikolaus Dietlein, Claudia Quedenau, Wei Chen, Sascha Sauer, Stephan Wolf, Thomas Höfer, Hans-Reimer Rodewald
Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system...
August 16, 2017: Nature
https://www.readbyqxmd.com/read/28811610/antibiotic-induced-release-of-small-extracellular-vesicles-exosomes-with-surface-associated-dna
#8
Andrea Németh, Norbert Orgovan, Barbara W Sódar, Xabier Osteikoetxea, Krisztina Pálóczi, Katalin É Szabó-Taylor, Krisztina V Vukman, Ágnes Kittel, Lilla Turiák, Zoltán Wiener, Sára Tóth, László Drahos, Károly Vékey, Robert Horvath, Edit I Buzás
Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells...
August 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28810059/targeted-genome-editing-in-caenorhabditis-elegans-using-crispr-cas9
#9
REVIEW
Behnom Farboud
Utilization of programmable nucleases to generate DNA lesions at precise endogenous sequences has transformed the ability to edit genomes from microbes to plants and animals. This is especially true in organisms that previously lacked the means to engineer precise genomic changes, like Caenorhabditis elegans. C. elegans is a 1 mm long free-living, nonparasitic, nematode worm, which is easily cultivated in a laboratory. Its detailed genetic map and relatively compact genome (~100 megabases) helped make it the first metazoan to have its entire genome sequenced...
August 15, 2017: Wiley Interdisciplinary Reviews. Developmental Biology
https://www.readbyqxmd.com/read/28804739/selection-of-genetically-modified-bacteriophages-using-the-crispr-cas-system
#10
Miriam Manor, Udi Qimron
We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous recombination with the plasmid, thus deleting the targeted gene. To select for these genomes, the CRISPR-Cas system is used to cleave non-edited genomes, enabling isolation of the desired recombinant phages...
August 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28802495/the-immunology-of-melanoma
#11
REVIEW
Jennifer S Ko
The relatively high DNA mutational burden in melanoma allows for the creation of potentially "foreign," immune-stimulating neoantigens, and leads to its exceptional immunogenicity. Brisk tumor-infiltrating lymphocytes, a marker of immune editing, confer improved overall survival in melanoma, possibly due to reduced sentinel lymph node spread. Meanwhile, T-cell-stimulating drugs, so-called T-cell checkpoint inhibitors, which reverse peripheral tolerance-dependent tumor escape, have demonstrated unparalleled clinical success in metastatic melanoma...
September 2017: Clinics in Laboratory Medicine
https://www.readbyqxmd.com/read/28800965/the-crispr-cas9-facilitated-multiplex-pathway-optimization-cfpo-technique-and-its-application-to-improve-the-escherichia-coli-xylose-utilization-pathway
#12
Xinna Zhu, Dongdong Zhao, Huanna Qiu, Feiyu Fan, Shuli Man, Changhao Bi, Xueli Zhang
One of the most important research subjects of metabolic engineering is the pursuit of balanced metabolic pathways, which requires the modulation of expression of many genes. However, simultaneously modulating multiple genes on the chromosome remains challenging in prokaryotic organisms, including the industrial workhorse - Escherichia coli. In this work, the CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique was developed to simultaneously modulate the expression of multiple genes on the chromosome...
August 9, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28800419/genome-editing-and-plant-transformation-of-solanaceous-food-crops
#13
REVIEW
Joyce Van Eck
During the past decade, the ability to alter plant genomes in a DNA site-specific manner was realized through availability of sequenced genomes and emergence of editing technologies based on complexes that guide endonucleases. Generation of targeted DNA breaks by ZFNs, TALENs, and CRISPR/Cas9, then mending by repair mechanisms, provides a valuable foundation for studies of gene function and trait modification. Genome editing has been successful in several food crops, including those belonging to the Solanaceae, which contains some of the most widely used, economically important ones such as tomato and potato...
August 8, 2017: Current Opinion in Biotechnology
https://www.readbyqxmd.com/read/28797648/overview-of-transcriptomic-analysis-of-all-human-proteases-non-proteolytic-homologs-and-inhibitors-organ-tissue-and-ovarian-cancer-cell-line-expression-profiling-of-the-human-protease-degradome-by-the-clip-chip%C3%A2-dna-microarray
#14
REVIEW
Reinhild Kappelhoff, Xose S Puente, Claire H Wilson, Arun Seth, Carlos Lopez-Otin, Christopher M Overall
The protease degradome is defined as the complete repertoire of proteases and inhibitors, and their nonfunctional homologs present in a cell, tissues or organism at any given time. We review the tissue distribution of virtually the entire degradome in 23 different human tissues and 6 ovarian cancer cell lines. To do so, we developed the CLIP-CHIP™, a custom microarray based on a 70-mer oligonucleotide platform, to specifically profile the transcripts of the entire repertoire of 473 active human proteases, 156 protease inhibitors and 92 non-proteolytically active homologs known at the design date using one specific oligonucleotide per transcript...
August 7, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28791391/a20-protein-regulates-lipopolysaccharide-induced-acute-lung-injury-by-downregulation-of-nf-%C3%AE%C2%BAb-and-macrophage-polarization-in-rats
#15
Ying Wang, Zhenju Song, Jing Bi, Jie Liu, Lin Tong, Yuanlin Song, Chunxue Bai, Xiaodan Zhu
Modulation of inflammation is a crucial component of the development of acute lung injury. A20, a ubiquitin editing enzyme, may regulate cellular inflammatory reactions, particularly those involving the signaling pathway of nuclear factor NF-κB (NF‑κB). The present study investigated the mechanism by which A20 downregulated NF‑κB and further contributed to macrophage polarization from the M1 to M2 phenotypes in lipopolysaccharide (LPS)‑induced lung injury. Sprague‑Dawley rats injected with LPS were used in the present study...
August 7, 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/28783728/correction-of-a-pathogenic-gene-mutation-in-human-embryos
#16
Hong Ma, Nuria Marti-Gutierrez, Sang-Wook Park, Jun Wu, Yeonmi Lee, Keiichiro Suzuki, Amy Koski, Dongmei Ji, Tomonari Hayama, Riffat Ahmed, Hayley Darby, Crystal Van Dyken, Ying Li, Eunju Kang, A-Reum Park, Daesik Kim, Sang-Tae Kim, Jianhui Gong, Ying Gu, Xun Xu, David Battaglia, Sacha A Krieg, David M Lee, Diana H Wu, Don P Wolf, Stephen B Heitner, Juan Carlos Izpisua Belmonte, Paula Amato, Jin-Soo Kim, Sanjiv Kaul, Shoukhrat Mitalipov
Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template...
August 2, 2017: Nature
https://www.readbyqxmd.com/read/28782493/big-data-and-genome-editing-technology-a-new-paradigm-of-cardiovascular-genomics
#17
Chayakrit Krittanawong, Tao Sun, Eyal Herzog
Cardiovascular diseases (CVDs) encompasse a range of conditions extending from congenital heart disease to acute coronary syndrome most of which are heterogenous in nature and some of them are multiple genetic loci. However, the pathogenesis of most CVDs remains incompletely understood. The advance in genome-editing technologies, an engineering process of DNA sequences at precise genomic locations, has enabled a new paradigm that human genome can be precisely modified to achieve a therapeutic effect. Genome-editing includes the correction of genetic variants that cause disease, the addition of therapeutic genes to specific sites in the genomic locations, and the removal of deleterious genes or genome sequences...
August 4, 2017: Current Cardiology Reviews
https://www.readbyqxmd.com/read/28781234/structural-basis-for-the-canonical-and-non-canonical-pam-recognition-by-crispr-cpf1
#18
Takashi Yamano, Bernd Zetsche, Ryuichiro Ishitani, Feng Zhang, Hiroshi Nishimasu, Osamu Nureki
The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM...
August 17, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28777443/use-of-human-pluripotent-stem-cell-derived-cardiomyocytes-to-study-drug-induced-cardiotoxicity
#19
Agnes Maillet, Kim Peng Tan, Liam R Brunham
Drug-induced cardiotoxicity is the one of the most common causes of drug withdrawal from market. A major barrier in managing the risk of drug-induced cardiotoxicity has been the lack of relevant models to study cardiac safety. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in drug discovery and cardiotoxcity screens as they display many characteristics of the human myocardium and offer unlimited supply. This unit describes how to use pluripotent stem cells derived cardiomyocytes to study drug-induced cardiotoxicty using doxorubicin as an example...
August 4, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28767689/crisprpred-a-flexible-and-efficient-tool-for-sgrnas-on-target-activity-prediction-in-crispr-cas9-systems
#20
Md Khaledur Rahman, M Sohel Rahman
The CRISPR/Cas9-sgRNA system has recently become a popular tool for genome editing and a very hot topic in the field of medical research. In this system, Cas9 protein is directed to a desired location for gene engineering and cleaves target DNA sequence which is complementary to a 20-nucleotide guide sequence found within the sgRNA. A lot of experimental efforts, ranging from in vivo selection to in silico modeling, have been made for efficient designing of sgRNAs in CRISPR/Cas9 system. In this article, we present a novel tool, called CRISPRpred, for efficient in silico prediction of sgRNAs on-target activity which is based on the applications of Support Vector Machine (SVM) model...
2017: PloS One
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