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We narrowed to 278 results for: guide rna expression plasmid

Showing: 41 - 60 of 278 results

  1. Viral Vectors 101: Parts of the AAV Transfer Plasmid

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    Blog Post
    ...with some plasmids. You just need three plasmids to start making AAV:  the packaging plasmid which contains... transfer plasmid an AAV transfer plasmid. ITR sequences are 145 bases each and AAV plasmids have two ...aids in the nuclear export of RNA and RNA translation, and promotes RNA transcript longevity. The polyA...of an AAV transfer plasmid. Parts shown in green are key elements of an AAV plasmid. Parts shown in grey... elements of an AAV plasmid First let’s talk about the key parts of an AAV plasmid. Inverted terminal ... of expression. For example, CAG is a strong promoter that drives very high, ubiquitous expression, while...transfer plasmids have a promoter, but this element isn’t required when the transfer plasmid harbors non-coding...

  2. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

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    Blog Post
    ...control RFP expression plasmid.  With this set up, all successfully transfected cells will express RFP while...using the supplied donor plasmid will express GFP. If there’s GFP expression, presumably the endogenous...fluorescent protein for assessing CRISPR/Cas9 activity Guide RNA structure and the site in the genome you are trying...Resources on Addgene.org Read our CRISPR guide Find CRISPR plasmids for your research Find resources and ... different gRNAs by simply changing the gRNA used when transfecting your cells.  A plasmid that contains...iSRIRACCHA) where Cas9 expression is activated using 4-OH-T and doxycycline. A gRNA that targets your desired...the N-terminus of mCherry can be targeted by a sgRNA guided Cas9 causing a DSB break. This DSB is repaired...

  3. Hot Plasmids: Spring 2025

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    Blog Post
    ...empty and EGFP retroviral plasmid set, our bacterial plasmid set for expressing tagged proteins, and stay...the payload (teal). The Cas9 gRNA targeting guide and the transposon RNA homology both help direct the...of the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles. ...backbones special: Strong expression: A CMV promoter drives robust gene expression in mammalian cells. Flexible...stay tuned for mammalian expression and AAV plasmid sets launching later this year!  Find Addgene's own...retrotransposons for DNA editing By Emily P. Bentley For RNA-guided genome editing, think beyond Cas9 with STITCHR...bioRxiv, under a CC-BY-NC-ND 4.0 International license. These plasmids are part of the BRAIN Initiative...

  4. CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9

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    Blog Post
    ...short ncRNAs, such as microRNAs that are around 20 base pairs long. Using a single guide RNA (sgRNA) to ...please… non-coding RNAs! You probably didn’t see that coming, right? Non-coding RNAs (ncRNAs) are transcribed...Characterization of Long Non-Coding RNAs. Non-Coding RNA. 2021;7(4):79. doi:10.3390/ncrna7040079 Horlbeck MA, Liu SJ,...CRISPR/Cas13, which targets RNA instead of DNA and selectively degrades RNA molecules. This system is often...CRISPR Guide Addgene's CRISPR 101 eBook CRISPR resources More resources on the Addgene blog RNA Editing...Editing with Cas13 Targeting RNA with Cas13a (C2c2) Cas13d: Small RNA-targeting CRISPR enzymes for transcriptome...Doudna JA, Charpentier E. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity...

  5. Hot Plasmids June 2018 - Reverse transcriptase, nanobody, and protein-DNA interaction tools

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    Blog Post
    ...transcription of RNA such as long-read cDNA sequencing and RNA sequencing of complex heterogeneous RNA samples....a sequence of interest with an sgRNA expressed from a separate plasmid. When cells are treated with biotin–phenol...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...write about a recent plasmid deposit please sign up here. Listen to the Hot Plasmids Podcast!  MarathonRT...at Yale University recently deposited a plasmid for expressing a novel reverse transcriptase (RT), MarathonRT...processivity. Please find the MarathonRT plasmid here! Zhao C, et al. RNA. 2018 PubMed PMID:29109157. Nanobody...

  6. CRISPR Activation: A Practical Guide

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    Blog Post
    ...three or four guides directed towards that gene as there can be a large difference in guide potency. Thus...2018). cDNA libraries, which consist of plasmids that over-express coding sequences from a given cell type...2015) WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system. Genome Biol 16: ....said, we generally pick guides by putting the promoter of the gene into a gRNA finder such as WU-CRISPR...or our lab’s sgRNA scorer 1.0 (Now 2.0) (Chari et al., 2015) and picking whichever guides are closest ...used in therapeutics, as it can increase gene expression at certain loci to create changes in phenotypes...: once the environmental trigger leaves, gene expression is no longer activated. However, there are several...

  7. What’s in Your Tube?: A Quick Guide to Measuring DNA by Spectrophotometry

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    Blog Post
    ...sample.  RNA contaminants DNA samples can also be contaminated with RNA (and vice-versa). Both RNA and DNA...gel. In the case of plasmids, the use of RNase treatment to degrade bacterial RNAs in most purification... at the presence of RNA contamination (Koetsier and Cantor, 2019). To confirm RNA in your DNA sample, ...A260/A230 ratios in RNA preparations on downstream applications. Qiagen Gene Expression Newsletter. Issue...Best Way to Elute and Store Your Plasmid DNA? Selecting Your Plasmid Purification Kit Resources on Addgene.org...protocols reduces the risk of RNA contamination — just be sure to check that your RNase has not expired (or been...You have just purified your plasmid and are ready to move on to your downstream application — but wait...

  8. Harnessing TnpB for Genome Editing: A Compact and Versatile Tool for Your Research

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    Blog Post
    ...mechanism. Small RNA elements called right-end RNA (reRNA) or omega RNA (ωRNA) guide the TnpB to its DNA...cloning the 20-nt guide RNA is identical for both vectors. Here, we provide a step-by-step guide for using a...addgene.org Genome Engineering Guide Plant Plasmids and Resources CRISPR Plasmids and Resources ...single guide RNA (sgRNA) used in Cas systems. The structure is also reminiscent of sgRNAs, with reRNA containing... choice of promoter for ωRNA (reRNA) expression (see Figure 1). Both plasmids contain a hygromycin resistance...nucleotide (nt) guide sequence that is complementary to the DNA target. The 20-nt guide region binds to...colonies, grow them, and extract plasmids. Confirm the correct guide insertion by Sanger sequencing using...

  9. Plasmids 101: Expressing shRNAs for RNA Interference

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    Blog Post
    ...small RNA molecules, such as: Micro-RNA (miRNA) Short hairpin RNA (shRNA) Short interfering RNA (siRNA... comparison of an shRNA plasmid sequence and an miRNA plasmid sequence. (A) Plasmid sequence from pLVUHshp53...experiments. shRNA plasmids typically use RNA polymerase III promoters, which transcribe small RNAs. The most...using plasmids The design of shRNA plasmids requires three important steps: Designing the shRNA sequence... as you might say. RNA interference (RNAi) is, as the name might suggest, an RNA-mediated gene silencing... type of small RNA and can occur via transfection or viral delivery of a plasmid. RNAi is an umbrella ...engineered to skip this first pri-miRNA step. These RNA duplexes bind to the RNA-induced silencing complex (...

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

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    Blog Post
    ...impaired Cas protein (dCas or Cas nickase)  a guide RNA that targets the complex of TadA and dCas to the...Get the basics of CRISPR in our CRISPR guide Find all plasmids for base editing in our collection Sign...introduced into cells as a plasmid or as mRNA constructs. In primary human cells mRNA delivery of ABEs resulted...base changes in cellular DNA and RNA dependent or independent of the gRNA used (Gaudelli et al., 2017). ... et al., 2020) was able to reduce off-target RNA and gRNA-dependent DNA editing while maintaining on-target...and 𝛽-thalassemia, can be mitigated with the expression of fetal hemoglobin. Previous studies have shown...gamma hemoglobin (HGB1/2) leads to continuous expression of fetal hemoglobin even in adult patients. In...

  11. Cpf1: A New Tool for CRISPR Genome Editing

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    Blog Post
    ...100 nt crRNA/tracrRNA hybrid guides needed for Cas9 function. Since both Cpf1 and its guide RNAs are smaller...our CRISPR guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease...Later work, like the adaptation of Cas13 to RNA targeting and RNA editing, has further shown the diversity...References 1. Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system."...at Addgene Find CRISPR plasmids at Addgene Read Addgene's CRISPR Resource Guide View Other CRISPR Blog...t require a tracrRNA, crRNA guides are only ~42 nt long. Direct synthesis of these crRNAs should be significantly...pattern and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage....

  12. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

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    ...which one can design and synthesize CRISPR guide RNAs (gRNAs) for genome editing in large-scale has led...multiplexed gRNA expression (Wong et al., 2016), in addition to the combinatorial expression of other genetic...pool of lentiviral plasmids. Multiple rounds of digestion and ligation result in plasmid libraries containing...screening in mammalian cells with a lentiviral CRISPR-guide RNA library." Nature biotechnology 32.3 (2014): 267... CRISPR Background with Our CRISPR Guide Pages Find CRISPR Plasmids Find More Tools for Combinatorial ...lentiviral plasmids containing one or more gRNAs. As each ligation reaction uses a pool of gRNAs as starting...containing combinations of different gRNAs in each plasmid. Each gRNA combination can be tracked and quantitatively...

  13. 27 Hot Plasmids from 2016

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    Blog Post
    ...nickase, and Cas9 activator expressing plasmids that can co-express 1-8 gRNAs. Ordon et al., 2016 tested...constitutively express Cas9 with a library of sgRNA expression vectors (containing ten guides against each...assembly to produce single plasmid targeting vectors that encode both a guide RNA transcript and a repair...lines expressing it. The Church lab fixed this issue by creating a new dCas9-VPR expression plasmid that...transfer vector for in-vivo expression and plasmids optimized for dCas9-VPR expression in drosophila and yeast...Neurosci 2016. PubMed PMID: 26950006 Plasmid ID Plasmid Name Plasmid Type 74309 pcDNA3.0-TRPV4-p2A-ferritin-p2A-mCherry... HEK293FT cells expressing a single gRNA targeting a single gene. The gRNA expressing cells were then ...

  14. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

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    Blog Post
    ...2008). 2. Transient expression of transgenes/shRNAs If you want your cells to express a protein or nucleic...leading to transient expression of gene products (Wu et al., 2004). Over time, the expression diminishes as ...if continuously expressed (Fu et al., 2013). Using IDLVs ensures transient expression without the risks...risks associated with prolonged expression. 3. Stable expression in non-dividing cells IDLVs can also be...integrase-deficient packaging plasmid instead of the standard packaging plasmid. For more details on how to...Cycle Quick Guide to All Things Lentivirus More resources on addgene.org Lentiviral Guide   Popular lentiviral...protein called reverse transcriptase to convert their RNA genomes into DNA to be integrated into the chromosome...

  15. Lentiviral CRISPR Libraries Enable Genome-Scale, Knockout Screening

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    Blog Post
    ...Sabatini and Eric Lander, comprise 73,171 guide RNAs (gRNAs) covering a total of 7,114 human genes. Unlike...Screening in Mammalian Cells with a Lentiviral CRISPR-Guide RNA Library. Koike-Yusa H, Li Y, Tan E-P, Del Castillo...then with the sgRNAs, and can result in a higher titer due to the smaller size of the plasmids. The Zhang..., targets 18,080 human genes with 64,751 unique guide sequences to enable both negative and positive selection... thousands of targets at once. That’s what our plasmids allow.” Wang’s library is designed to target a... a smaller set of genes and it includes more guides per gene. The Sabatini/Lander library uses sequential...Zhang lab, on the other hand, uses a 1 plasmid, single infection system. Genome wide mouse CRISPR library...

  16. Giving gRNAs a Facelift - Synthetic and Beyond

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    Blog Post
    ...a ~20 bp gRNA which is part of the larger CRISPR RNA (crRNA). In addition to the 20 bp guide sequence,... when scientists talk about their precious RNA samples. RNA is inherently less stable than most macromolecules... as they will be recognized as foreign RNA. To protect the RNA from degradation, several simple methods...Bak, R., Clark, J. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary...M., Hendel, A. Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian... either the crRNA or the tracrRNA. Of course, in a single guide system (crRNA and tracrRNA as a single... trans-activating crRNA (tracrRNA) which interacts with the Cas9 protein. These RNA molecules are vulnerable...

  17. Viral Vectors 101: Gamma-Retroviral Packaging Systems

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    Blog Post
    ...γ-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G (Vesicular...in trans by specific plasmids. This method, therefore, reduces the number of plasmids that are required ...encoded by the transfer plasmid, but instead are provided in trans by other plasmids or the packaging cell...do many γ-retroviral plasmids have similar names? The nomenclature of the plasmid backbone is meant to...transduction efficiency and transgene expression. Psi (Ψ) RNA target site for packaging by Nucleocapsid...retro/lentiviral plasmids. U5; Unique 5'; region at the 5' end of the viral genomic RNA (but found at both...needed: γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest: The transgene sequence...

  18. A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression

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    Blog Post
    ...analyzing the plasmid and viral DNA sequences and expression profile at multiple steps from plasmid amplification... require gene expression in a specific cell type rely on transgenic models that express recombinases like...level of off-target transgene expression, low levels of spurious expression were still observed in many ...Causes of recombination and leaky expression While leaky expression and spontaneous recombination is a...recombination that can lead to leaky expression in cells that do not express Cre or Flp. Here at Addgene we ...off-target expression. Figure created with BioRender.com   Strategies for optimizing expression So now...DIO/Flex vectors and leaky expression can occur in cells that don’t express Cre or Flp, what can we do...

  19. Viral Vectors 101: Viral Vector Elements

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    Blog Post
    ...lentiviral plasmids Addgene's most popular retroviral plasmids Addgene's collection of AAV plasmids Addgene...split across several plasmids (often three), for safety purposes. The primary plasmid with the delivered...often a gene) is called the transfer plasmid while the other plasmids with packaging or replication components...similar, and is contained on the transfer plasmid. If expression is the goal, a promoter should flank this...what will be delivered and expressed in the host cell from the transfer plasmid. Viral packaging and entry...of adenovirus plasmids Resources on the Addgene blog Parts of the AAV packaging plasmid Overview of Lentiviral...cargo, typically an expression construct, to cells/organisms. This is often cDNA, shRNA, or something similar...

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

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    Blog Post
    ... a GAL-4 based bipartite expression system that can regulate gene expression in C.elegans. In this system...and plasmid assembly line for the creation of CRISPR targeting vectors that each contains a guide RNA ... CRISPR plasmid can be created in a single tube. You can also find additional SapTrap plasmids used for...about C. elegans and the plasmids we have at Addgene check out our worm expression resources page. This page...elegans Browse our Plasmids 101 blog posts Resources on Addgene.org Visit our worm plasmids and resources...individually depleted via RNA interference (RNAi). Scientists monitor the effects of RNAi using four-dimensional...CRISPR techniques and new tools for conditional expression and degradation. These workshops not only highlighted...
Showing: 41 - 60 of 278 results