Podcasts about nuclease

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.24.525105v1?rss=1 Authors: Sastre, D., Zafar, F., Torres, C. A. M., Piper, D., Kirik, D., Sanders, L. H., Qi, S., Schuele, B. Abstract: Parkinson's disease (PD) is one of the most common neurodegenerative diseases, but no disease-modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.29.226639v1?rss=1 Authors: Jinks-Robertson, S., Gamble, D., Shaltz, S. Abstract: Mitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae. Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3' overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol {delta}-mediated removal of a mismatch 8 nucleotides from a 3' end affects recombination outcomes and whether the presence of a 3' versus 5' overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers versus noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3' end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3' versus 5' overhangs differs, we compared the well-studied 3' overhang created by I-SceI to a 5' overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN- than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate. Copy rights belong to original authors. Visit the link for more info

Sam Sternberg discusses his work on exploring and exploiting CRISPR-Cas immune systems, beginning as a graduate student with Jennifer Doudna, at a biotech start-up, and in his laboratory at Columbia University. Host: Vincent Racaniello Guest: Sam Sternberg  Become a Patron of TWiM! Links for this episode Sternberg Laboratory at Columbia Mechanism of substrate selection by Cas9 (RNA) DNA interrogation by Cas9 (Nature) Conformational control of DNA target cleavage by Cas9 (Nature) A Crack in Creation by Doudna and Sternberg What if we could rewrite the human genome? (YouTube) Sam Sternberg Music used on TWiM is composed and performed by Ronald Jenkees and used with permission. Send your microbiology questions and comments to twim@microbe.tv

For more information, please visit: http://bitesizebio.com/webinar/25961/deciphering-steps-of-mrnp-assembly-in-developing-oocytes-using-super-resolution-microscopy/ All mRNA molecules recruit specific proteins to form ribonucleoprotein complexes (mRNPs). Composition and localization of many mRNPs change dynamically from translation to decay. Microscopic techniques with high spatial and temporal resolution are invaluable for studying mRNP biogenesis. We have developed new tools based on fluorogenic forced intercalation (FIT) probes for RNA detection, quantification and interference in biological samples. The probes contain a thiazole orange (TO) dye introduced at a position normally occupied by a nucleobase. Upon binding to target nucleic acids, the TO dye increases its quantum yield and brightness substantially (greater than10 fold). These probes detect mRNA in a rapid, wash-free FISH setup using conventional wide-field microscopy. It is an ideal tool for RNA localization screens. Nuclease resistant FIT probes containing a locked nucleic acid adjacent to the TO dye are bright and contrasted enough for use in live imaging. These probes can also be designed to target functional elements of RNAs to test the role of those in RNP biogenesis. Absorption and emission spectra of TO are sufficiently different from EGFP to enable high sensitivity and specificity RNA-protein co-localization analysis, even with super-resolution, to study the RNA interactome. LNA modified FIT probes are excellent subjects for STED microscopy as duplex formation greatly increases their fluorescence lifetime.

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, and Kathy Spindler Vincent, Dickson, Alan, and Kathy discuss disruption of the ccr5 gene in lymphocytes of patients infected with HIV-1. Links for this episode Gene editing of ccr5 in AIDS patients (NEJM) HIV gets the zinc finger (TWiV 144) Genome engineering with zinc finger nucleases (Genetics) Photo credit: Watty's Wall Stuff Mice lie, monkeys exaggerate t-shirt design (thanks, Christophe) Letters read on TWiV 278 Weekly Science Picks Alan - Digital scale model of solar systemDickson - Font of knowledgeVincent - Measles outbreaks trends, and NYC measles (one, two)Kathy - Winner, funding basic science to revolutionize medicine Listener Pick of the Week Stephen & Jon - Watty's Wall StuffJohyne - Macro views of snowflakesRicardo & Stephen - Vaccine exemptionsBill - Books by John JanovyMarshall - Animation of DNA replicationSteve - Debunking influenza vaccine myths Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

Hosts: Vincent Racaniello and Dickson Despommier Vincent and Dickson meet with Judith Straimer and Marcus Lee to discuss their method for site-specific genome editing in Plasmodium falciparumusing zinc finger nucleases. Right-click to download TWiP #46 (46 MB .mp3, 64 minutes). Links for this episode: Judith Straimer and Marcus Lee Genome editing in P. falciparum with zinc finger nucleases (Nature Methods) Zinc finger nucleases (Sigma-Aldrich) Gene-editing nucleases (Nature Methods) Illustration by Andrew Lee Contact Send your questions and comments (email or mp3 file) to twip@twiv.tv.

Sat, 21 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14607/ https://edoc.ub.uni-muenchen.de/14607/1/Fezert_Pauline.pdf Fezert, Pauline ddc:590, ddc:500, Tierärztliche Fakult

Vincent, Rich, and Alan discuss live blogging of scientific meetings, the current outbreak of Hendra virus is Australia, and using zinc finger nucleases to make HIV-resistant CD4 cells.

Fri, 1 Jan 1982 12:00:00 +0100 https://epub.ub.uni-muenchen.de/8803/1/bacteriophage_t4-induced_anticodon-loop_nuclease_detected_in_a_host_strain_restrictive_to_rna_ligase_mutants_8803.pdf Kaufmann, G.; Borasio, Gian Domenico; David, M.