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We narrowed to 345 results for: Ada

Showing: 41 - 80 of 345 results

  2. dTAG - You're it!

    Type
    Blog Post
    ...Dana-Farber Cancer Institute. Targeted protein degradation In the Bradner and Gray labs, we synthesize compounds...small molecule degraders that achieve selective degradation of endogenous proteins (notably, BRD2/3/4, CDK9... Hippel-Lindau (VHL) to bring the endogenous degradation machinery into close proximity with the target...polyubiquitination of the target protein and proteasomal degradation. Remarkably, small molecule degraders provide...prolonged biological effects. While targeted degradation of endogenous proteins is an exciting strategy...tag-based strategy, which we refer to as the degradation tag (dTAG) system, to co-opt the endogenous ubiquitin... evaluate the effects of rapid and selective degradation of protein targets in time scales that are not...

  3. Western Blot

    Type
    Protocol
    ...protocol uses a dry transfer device but can be adapted for wet and semi-dry transfer approaches. Last ...

  4. CRISPR 101: RNA Editing with Cas13

    Type
    Blog Post
    ...editing portion of the protein, they examined ADAR1 and ADAR2, which deaminate adenosine to inosine in RNA... functional A->G change. They fused ADAR deaminase domains (ADARDD) to dPspCas13b, but observed low RNA... editing, they employed hyperactive ADAR constructs, like ADAR2DD(E488Q). They also adjusted the structure...RNA. Using an ADAR2DD catalytic mutant, they showed that editing is mediated by ADAR2DD, not PspCas13b...tested truncated ADARDDs to see if they could shrink the construct. They identified ADAR2DD(delta984-1090...mutating residues in ADAR2 that interact with duplex RNA, they hoped to destabilize ADAR-RNA binding to decrease..., 2016) as a RNA-targeting CRISPR enzyme, they adapted Cas13b for precise RNA editing (Cox et al., 2017...

  5. Exploring Applications of the Bioluminescent HiBiT Tag

    Type
    Blog Post
    ...confirm efficient EV loading of ADAR1-HiBiT. When EVs from cells expressing ADAR1-HiBiT were added to LgBiT-...flow via EVs. Find ADAR-HiBiT Fusion Vector at Addgene! Watching protein degradation in real time Proteins...conducted by Kamada et al. (2023) used HiBiT to characterize the proteasomal degradation of cystic fibrosis...ubiquitin ligases in the degradation of CFTR, specifically UBE3C. Degradation of CFTR was measured through...components of the degradation pathway. HiBiT enabled the authors to observe degradation as well as endocytic...https://doi.org/10.1021/cb3002478 Kamada, Y., Tateishi, H., Nakayamada, U., Hinata, D., Iwasaki, A., Zhu...analyzing real-time kinetics, such as binding, degradation, trafficking (Hall et al., 2012). Antibody-based...

  6. Plasmids 101: Degron Tags

    Type
    Blog Post
    ...leading to ubiquitination and proteasomal degradation. Adapted from Nabet et al., (2018).   HaloTag...of a protein and can inducibly facilitate the degradation of the tagged protein. Degrons are destabilizing...various enzymes and sent to the proteasome for degradation. The tags we will discuss here are all conditional...conditional, meaning that they only facilitate degradation of their fused protein under certain conditions...are reversible mechanisms of control. Protein degradation can be induced by the addition of a small molecule...to normal once the drug has been removed. The degradation and reversal are rapid compared to other systems... entire tagged protein to the proteasome for degradation. In addition to having the coolest name of all...

  7. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    ...NNNRRT 8 ABE SpCas9 TadA-TadA* NGG 9-12 SpCas9-VQR TadA-TadA* NGA 8 SaCas9 TadA-TadA* NNGRRT 9 SaCas9...SaCas9-KKH TadA-TadA* NNNRRT 8 SpCas9-VRER TadA-TadA* NGCG 8  Tips for designing base editing experiments...evolution and engineering gave rise to a mutant TadA (TadA*) that satisfactorily accepts DNA as its substrate... of different basic BE technologies and their adaptation for plant genome editing. Cytosine base editors...mutant library of E. coli tRNA adenosine deaminases (ecTadA), fused it with dCas9, and transformed it into ... at The Pennsylvania State University (PSU) is adapting and improving base editing platforms for plant...editing using hybrid prokaryotic and vertebrate adaptive immune systems." Science 353.6305 (2016): aaf8729...

  8. A New Generation of Adenine Base Editors Improves Editing in Primary Human Cells

    Type
    Blog Post
    ...evolved TadA domain. Because ABE7.10 contains a heterodimeric fusion of wild-type TadA and TadA7.10, they...wild-type TadA and the evolved TadA region. The ABE8.x-m version contains only the evolved TadA and is ~...3 components:  a deaminase (TadA, originally from E.coli, named TadA7.10 in ABE7.10)  a catalytically...nickase)  a guide RNA that targets the complex of TadA and dCas to the genomic DNA of interest  ...When ABEs are in action they bind DNA so that the TadA domain can edit the short single-stranded DNA stretch...2017) in combination with a synthetic library of TadA sequences to create the eighth generation of adenine...antibiotic selection. As a result, 8 mutations in TadA could create all three edits and were thus more ...

  10. Expanding the Targeting Scope and Editing Efficiency of Adenine Base Editors

    Type
    Blog Post
    ...testing numerous TadA variants, they identified TadA-8e, which edits 590-fold faster than TadA-7.10. It is ...deoxyadenosine deaminase, TadA-7.10 (Gaudelli et al., 2017). The idea was to evolve a TadA-7.10 version with ...evolution of TadA variants with much higher base editing activity compared to the original TadA-7.10 enzyme...additional mutation (V106W) into TadA-8e and the resulting ABE8e(TadA-8e V106W) was as efficient in on-target...codons within T7 RNAP. A phage encoding the deaminase TadA-7.10 A mutagenesis plasmid controlling the phage...

  11. Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

    Type
    Blog Post
    ...assays Long-lived protein degradation: This approach measures the degradation rate of radio-labeled long-lived...this is indicative of protein degradation). To control for protein degradation due to other pathways, it ...cytosol and organelles to the vacuole/lysosome for degradation and recycling. The key to Rosella’s autophagy-sensing...short-lived proteins primarily via proteasome degradation. Autophagy is then induced and quantified by ...it is standard practice to compare degradation rates between samples treated with or without an autophagy... measure of autophagy, but, like the protein degradation assay above, it’s slow and only measures bulk...because their contents can be at various stages of degradation. Additionally, electron microscopy analysis is...

  12. CRISPR Library Amplification

    Type
    Protocol
    ...adequate quality, but this upfront cost will save headaches and expense later. At a minimum we recommend the...

  13. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    ...systems to generate ABE8e(TadA-8e V106W), which edits ~590-fold faster than the TadA from ABE7.10 without ...Engineered TadA domain for cytosine base editing Lam et al., 2023 CBE-T, CABE-T   Engineered TadA domain...evolution to create one from the RNA adenine deaminase TadA.   After seven rounds of molecular evolution, they...approach, engineering and directed evolution of the TadA domain has yielded base editors that are equally... Neugebauer et al., 2023 TadCBE, TadDE   Evolved TadA domain for cytosine base editing (TadCBE) or dual...al., 2025 TadCBE(NCN) variants, TadCABE   Evolved TadA domain for position-specific cytosine base editing... D. (2023). Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine...

  14. Meet the New Addgene Board Members!

    Type
    Blog Post
    ...members and get to know them! Khandan Baradaran, PhD Khandan Baradaran’s expertise in viral safety, gene therapy... with health authority regulations,” Baradaran says.  Baradaran’s path to science began in high school...new members to the Board of Directors: Khandan Baradaran, Frank Stegmeier, and Luk Vandenberghe. We’re ...in viral genetics. In regards to open science, Baradaran thinks the best way to solve problems is to work...think will get the research funded,” she says. Baradaran met Addgene Executive Director Joanne Kamens several...

  15. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    ...binding and subsequent DNA degradation appears to be conserved. This DNA degradation portion can be bypassed.... These alterations are achieved through DNA degradation, thanks to the unique capabilities of Cas3 and...of DNA, Cascade caught up to it, and then DNA degradation continued. However, Cas3 cannot bind DNA on its...Hopefully more people will want to utilize Cas3’s degradative power in the future! References and Resources.... (2018). Structure basis for RNA-guided DNA degradation by Cascade and Cas3. Science, 361(6397). https...., Sonpho, E., Xu, H., Sasakawa, N., Naito, Y., Nakada, S., Yamamoto, T., Sano, S., Hotta, A., Takeda,...

  16. Rewiring Metabolic Circuitry with CRISPR RNA Scaffolds [Video]

    Type
    Blog Post
    ...contributed by Adam Chin-Fatt, a Ph.D. student at the University of Western Ontario. Adam summarizes Zalatan...Transcriptional Programs with CRISPR RNA Scaffolds." Adam has also created a video to help scientists visualize...and flexible technique that it should be on the radar of every molecular microbiologist. Summary  The...transcriptional programs. Thank you to our guest blogger! Adam Chin-Fatt is a Ph.D. student at the University of...recombinant pharmaceuticals. Follow him on Twitter @AdamChinFatt     References Qi, L. S. et al. Repurposing...

  17. Protein Tagging with CRISPR/Cas9: A Conversation with Mendenhall and Myers

    Type
    Blog Post
    ...more quickly and efficiently, they’ve recently adapted the CRISPR/Cas9 system for protein tagging. We .../crispr/tagging. Addgene: What motivated you to adapt CRISPR for use in protein tagging? Myers: Our labs...transcription factors. Our groups got together to adapt CRISPR for tagging experiments and it’s already ... improvements in efficiency and throughput. In adapting the CRISPR/Cas system, what we are doing is adding... is so flexible. Other investigators can easily adapt these vectors for use with their favorite selection...who’ve developed their own system or who use and adapt our reagents to think about adding them to Addgene...

  18. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
    ... Fig. 3 dTag mechanism of protein degradation. Adapted from Nabet et al. Check out Addgene's...with a degron tag can provide inducible protein degradation. These systems work by tagging the endogenously...mark the tag and whatever it is fused to for degradation. Since the tagged protein of interest is normally...off (SMASh), auxin-inducible degron (AID), and degradation tag (dTAG). These control systems function at...system for immediate and target-specific protein degradation. Nature Chemical Biology. May 2018. 14(5): 431...

  19. Science Communication for Everyone

    Type
    Blog Post
    ...post was contributed by guest blogger Chinmaya Sadangi, a postdoc at the University of Toronto. The Addictive...communicating science to non-scientists. Chinmaya Sadangi, a postdoc at the University of Toronto, created...underrepresented minorities to study STEM subjects. Here, Sadangi and his team answer a few questions about The Addictive...guest blogger, Chinmaya Sandangi! Dr. Chinmaya Sadangi is currently a postdoc at the University of Toronto...Germany in 2017. Apart from The Addictive Brain, Dr. Sadangi is an eLife ambassador, SfN community leader, and...

  20. Hot Plasmids and Viral Preps - March 2021

    Type
    Blog Post
    ... leading to the degradation of the AID-tagged protein. To quantify protein degradation the Ward lab used... that allows for both spatiotemporal protein degradation and quantification of this depletion using imaging-based... inducible degron sequence) tagged proteins. Degradation of your protein interest would also result in...system that brings Cas9 to ubiquitin ligase for degradation.      New from the viral service By Jason ...

  21. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    ...acids at the atomolar level. The Zhang lab has adapted natural RNase activity of the Cas13 protein to ...Doudna lab has used Cas12a’s non-specific ssDNA degradation to develop the method termed DNA Endonuclease...DETECTR harness the promiscuous cleavage and degradation of neighboring ssRNA and ssDNA by Cas13 and Cas12a...detection signal for a single target. SHERLOCKv2 was adapted so that a cleaved reporter could be detected on...built upon. Mammoth Biosciences and UCSF recently adapted the DETECTR platform to detect SARS-CoV-2 using...

  22. New Optogenetic Tools for Cytoskeleton and Membrane Control

    Type
    Blog Post
    ...Schematic of Opto-katanin’s use in cells. Images adapted from Meiring et al., 2022, under CC-BY 4.0 license...blue light illumination. Scale bar 25 µm. Images adapted from Mahlandt et al., 2023, under CC-BY 4.0 license...vesicle trafficking via CRY2-CIB1 clustering. Image adapted from Aljiboury et al., 2023, under CC-BY 4.0 license...colocalizing with SuperPLDx30. Scale bar 10 µm. Images adapted from Tei et al. 2022, bioRxiv, under a CC-BY-NC-ND...doi.org/10.1371/journal.pgen.1010765.  Berlew, E. E., Yamada, K., Kuznetsov, I. A., Rand, E. A., Ochs, C. C....

  23. With an Eye Towards the Future, We Look Back at the March for Science

    Type
    Blog Post
    ... on human artificial chromosomes. Montreal, QC, Canada 45.5017°N 73.5673°W – Vanessa Sung Hundreds of...de-funding of crucial environmental research programs, Canada has been here before.  These attacks on science...Research Centre at McGill University in Montreal, Canada.  Outside the lab, she dabbles in science policy...Intellectual Ventures Laboratory. Vancouver, BC Canada 49.2827°N 123.1207°W – Karissa Milbury I attended...University of British Columbia in Vancouver, BC (Canada). She works in a lab that uses yeast cells to study...

  24. Typing CRISPR Systems

    Type
    Blog Post
    ... all CRISPR types; the fast evolution of these adaptive bacterial and archaeal immune systems has led ...suspected that they don’t carry out the same types of adaptive immune functions that other CRISPR types do, due...resembles cas10. All type IV subtypes are missing adaptation genes, Cas1 and Cas2, which are critical for ...pyogenes) belonging in II-A. Cas9 was the first to be adapted for use in mammalian systems, with the landmark... become the most engineered Cas enzyme, with adaptations for increased fidelity, activation/repression...

  25. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...protocols, visit https://clover.caltech.edu. Figure adapted from Challis et al. 2022. Image created with BioRender.com...New World Monkeys and rodents. Image and caption adapted from Campos et al., 2023. AAV.CAP-Mac is a...doi.org/10.1038/nn.4593 Chen, X., Ravindra Kumar, S., Adams, C. D., Yang, D., Wang, T., Wolfe, D. A., Arokiaraj...Zheng, Q., Beddow, T., Zhao, B., Tobey, I. G., Moncada-Reid, C., Eid, F.-E., Walkey, C. J., Ljungberg,...Konno, A., Mochizuki, R., Shinohara, Y., Nitta, K., Okada, Y., & Hirai, H. (2018). Intravenous administration...Somasundaram, S., Miller, J. A., Kalmbach, B. E., Radaelli, C., Gore, B. B., Weed, N., Omstead, V., Bishaw...Widrick, J. J., Messemer, K. A., Troiano, E. C., Moghadaszadeh, B., Peacker, B. L., Leacock, K. A., Horwitz...

  26. What's the Best Way to Elute and Store Your Plasmid DNA?

    Type
    Blog Post
    ...product, or chromosome. One of the main causes of degradation during plasmid DNA extraction and purification...protein endonuclease I encoded by the gene endA. Nowadays, the most common E. coli strains used for plasmid... extra steps like heat inactivation, protein degradation, and/or the use of chelating agents that can ...store your sample without having substantial degradation and which experiments you can use your plasmid...

  27. The Open Repository of CRISPR Screens: CRISPR Screen Data in One Place

    Type
    Blog Post
    ...relevant metadata and the screen results. Screenshots updated 2024. Importantly, metadata for each...gene hits for each screen are also captured as metadata.   Screen results are reported as the original... diversify, the BioGRID ORCS resource will be adaptable to a wide variety of screen formats and contexts...

  28. New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor

    Type
    Blog Post
    ...SF-Venus-iGluSnFR.A184V, iGluSnFR3 v82, and iGluSnFR3 v857. Image adapted from Aggarwal et al., Figure 1   Increased...curves for the different iGluSnFR variants. Image adapted from Aggarwal et al…, Supplementary Figure 3. .../F0 for the different iGluSnFR variants. Image adapted from Aggarwal et al…, Supplementary Figure 2. ... PDGFR, GPI, and SGZ display constructs. Image adapted from Aggarwal et al… Figure 4. PDGFR is a C-terminal...

  29. Open Resources and Plasmid Tools For Studying C. elegans

    Type
    Blog Post
    ...for the Auxin Inducible Degradation System (AID) The Dernberg lab in 2015 adapted the AID system discovered...and new tools for conditional expression and degradation. These workshops not only highlighted new tools...  Zhang, Liangyu, et al. "The auxin-inducible degradation (AID) system enables versatile conditional protein...

  30. Design Tips for Prime Editing

    Type
    Blog Post
    ... motifs at their 3′ end to protect them from degradation. When designing epegRNAs with large structured...different strategies for protecting pegRNAs from degradation. Created with BioRender.com. That’s all ...A., Sahin, M., Osborn, M. J., Weissman, J. S., Adamson, B., & Liu, D. R. (2021). Enhanced prime editing...., Bauer, D. E., Marson, A., Parsons, L. R., & Adamson, B. (2024). Improving prime editing with an endogenous...

  31. Hot Plasmids: Winter 2025

    Type
    Blog Post
    ...weeks after herbicide was applied to both plants. Adapted from Vu et al., 2024 bioRxiv, under CC-BY-NC-ND... B) Protein expression and labeling procedure. Adapted from Smith et al. 2024 under a CC-BY 4.0 license...(C) is indicated; red line is exponential fit. Adapted with permission from Hao and Lee et al., 2024. ...non-neurodegenerative diseases (Diabetes & cancer). Image adapted from Wu and Wang 2024 under a CC-BY 4.0 license...

  32. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    ...purification of a specific genomic region, numerous adaptations of this system have been made in different fields...specific genomic region has also recently been adapted to isolate genomic loci in bacteria (Fujita et ...also has great potential for improvements and adaptations that can provide an increased understanding of...programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity." Science337.6096 (2012): 816...

  33. Using AAV for Neuronal Tracing

    Type
    Blog Post
    ...endocytosed material or molecules targeted for degradation. These two pathways also use different cytoskeletal...several parallel mechanisms in place (for review see Maday et al., 2014). It is currently unclear which proteins...Common AAV Serotypes and their target tissues (adapted from Addgene's AAV guide)  Commonly used serotypes...PMID: 22196330. PubMed Central PMCID: PMC3275419. Maday, S., Twelvetrees, A.E., Moughamian, A.J., and Holzbaur...PubMed Central PMCID: PMC4269290. Masamizu, Y., Okada, T., Kawasaki, K., Ishibashi, H., Yuasa, S., Takeda...

  34. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    .../Cas9 for gene editing has expanded since its adaptation for use in mammalian cells in 2012-2013. Researchers... gene (although it has been quite successfully adapted for use in screening experiments as well). Therefore...fluorescence indicates a more successful gRNA design. Adapted from Mashiko et al., 2014. Not all gRNA’s are...chance of contamination with RNase leading to degradation that can set you back days. Working with plasmid...

  35. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    ...immune system. The techniques used here could be adapted to studying other systems in which there is large... revealing the cellular behaviors that mediate adaptive and innate immunity in diverse tissue environments... to better understand T cell initiation in the adaptive immune response. It was known that T cells were...scale when using photoactivatable FPs - their degradation within labelled cells limits the timespan available...

  36. Special Delivery: Fluorophore Targeting for FRET Studies

    Type
    Blog Post
    ...developed by Roger Tsien and co-workers (Griffin, Adams, & Tsien, 1998), these dyes are now commercially...loops are best since these tags form beta-hairpins (Madani et al., 2009). Donor and acceptors: Putting it ...PMID: 19823671. 5. Griffin, B. Albert, Stephen R. Adams, and Roger Y. Tsien. "Specific covalent labeling...12125. Journal of the American Chemical Society. 7. Madani, Fatemeh, et al. "Hairpin structure of a biarsenical...

  37. The 12 Days of CRISPR

    Type
    Blog Post
    ... On the 10th day of CRISPR, Addgene gave to me: RADARS from Gootenberg, Chen, and Abudayyeh🎵On the 9th...9th day of CRISPR, Addgene gave to me: RADAR from Gao🎵On the 8th day of CRISPR, Addgene gave to me: Functional...High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects🎵 On the 1st ...

  39. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    ...Traditional sgRNAs are protected from cellular degradation by Cas9 binding, but pegRNAs have an extended... RNA stability This recent contribution by the Adamson lab also addresses the problem of RNA stability...A., Sahin, M., Osborn, M. J., Weissman, J. S., Adamson, B., & Liu, D. R. (2021). Enhanced prime editing...., Bauer, D. E., Marson, A., Parsons, L. R., & Adamson, B. (2024). Improving prime editing with an endogenous...

  40. What's New in CRISPR - December 2019

    Type
    Blog Post
    ...AID) and an engineered tRNA adenosine deaminase (TadA) to a catalytically impaired Streptococcus pyogenes...Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Now, they’ve further developed this editor for ...editing by evolving the adenine deaminase domain of ADAR2 into a cytidine deaminase. This editor still retains...
Showing: 41 - 80 of 345 results