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We narrowed to 278 results for: guide rna expression plasmid

Showing: 81 - 100 of 278 results

  1. Introducing an All-in-One CRISPR/Cas9 Vector System for Multiplex Genome Engineering

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    Blog Post
    ...all-in-one CRISPR/Cas9 vectors expressing up to seven guide RNAs (gRNAs). Sakuma said that the new system...Golden Gate cloning method to ligate expression cassettes of the gRNAs into a single vector in tandem. They...on earlier evidence showing that a CRISPR/Cas9 plasmid could be injected directly into mice for genome...These are sure to be powerful tools to modify the expression of multiple genes at the same time.” For more... to Design Your gRNA for CRISPR Genome Editing CRISPR-Cas9: Tips for Optimizing sgRNA Activity Lentiviral...

  2. Viral Vectors 101: Types of viruses

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    Blog Post
    ...Addgene.org Adeno-associated Virus (AAV) Guide Addgene’s viral plasmids and resources Addgene’s viral services...Gamma-retrovirus Retroviruses are single-stranded RNA viruses that use reverse transcriptase to synthesize...integrate into proto-oncogenes and disrupt their expression (Robinson, 1982). To add insult to injury, retroviruses...their required components are very similar. While plasmids containing virus-specific elements such as the...integrate, and the addition of a helper virus or plasmid allows it to replicate episomally, essentially ...cells and doesn’t require genomic integration to express its cargo, in fact its viral life cycle does not...not include genomic integration. While the expression of adenovirus cargo is high, it’s generally not ...

  3. Your Top Requested Plasmid in 2016!

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    Blog Post
    ...versatile mammalian expression vector that you can use to express both SpCas9 and your gRNA in a variety of...versatile mammalian expression vector that you can use to express both SpCas9 and your gRNA of interest in ...Similar to last year, the top requested plasmid from 2016 is an expression vector for CRISPR nuclease, SpCas9...mammalian cell types. Purpose  SpCas9 This plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh... CRISPR Plasmids to Mammalian Cells Resources on the Addgene Website Visit our CRISPR Guide Pages Browse...NGG3' PAM sequence as directed by a gRNA. 2A-Puro In this plasmid, SpCas9 is fused to the puromycin resistance... clone your 20 nucleotide guide sequence of choice (for generation of a gRNA that will target SpCas9 to...

  4. Delivery Methods for Generating iPSCs

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    Blog Post
    ... as a donor plasmid containing the transposon, along with a helper plasmid that expresses the transposase...lingering expression of reprogramming factors. Non-replicating Plasmids: Generating iPSCs with plasmid-based...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors...reprogramming. Additionally, lingering expression or re-expression of viral transgenes in iPSC-derived cells... generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...the dose of plasmid the cells receive over the whole reprogramming period and the plasmid will get diluted...larger plasmids have lower rates of transfection. Minicircles: Minicircles are like mini-plasmids. They...

  5. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

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    Blog Post
    ...differences in guide RNA efficiency. For example, in a two-guide floxed experiment, if one guide cleaves less...CRISPR Guide Find Plasmids for Cas9 Purification Find CRISPR Plasmids for Mammalian Expression ...Cas9 protein and the separated guide RNA components (crRNA and tracrRNA) for maximum Easi-CRISPR efficiency...successfully used guides targeting the same or complementary strands, as well as guides facing towards or...editing. How to use Addgene plasmids for Easi-CRISPR You can use Addgene plasmids to create a repair template...of Addgene plasmid options for templates - see the example at right using blue flame plasmid pmScarlet_...desired insert (e.g., mScarlet).   Plasmid ID Plasmid Name Tags 69090 pMJ915 MBP (N ...

  6. Antibody Validation for Flow Cytometry

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    Blog Post
    ...flow cytometry, and mRNA expression.   If your protein of interest is expressed at similar levels in...epitopes. Detection of overexpressed (tagged or untagged) protein When expression plasmids are available, or...confirmation of RNA knockdown by RT-qPCR, and potentially the need to study protein expression at multiple...Correlation with RNA or Proteomic data from multiple cell lines/ types with different expression Flow cytometry... involves the transient transfection of expression plasmids in a given cell line. For flow cytometry, ...useful to produce a sample with mixed expression, with the expression measurable through an epitope tag such...studies are not feasible, an alternative approach is RNA interference (RNAi). The most common method is...

  7. CrispyCrunch: High-throughput Design and Analysis of CRISPR+HDR Experiments

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    Blog Post
    ... proteins. For each gene, we needed to design guide RNAs, donor templates and sequencing primers for quality...mutating guide sequences in a way that prevents re-cutting but does not disrupt gene expression. (See section... section below.) For guide design, we decided to adapt the popular guide design tool Crispor by Max Haeussler...return guides and primers in batch, optimized for HDR. Compared to wealth of resources for guide design... summary per batch. Ranking guides for HDR in-depth In other tools, guides are ranked by specificity of...specificity of a guide to the target. The CFD score ranges between 0 and 100 for each guide, with 100 being... the guide and the target and 0 being the weakest interaction due to mismatches between the guide and ...

  8. Antibodies 101: Validation

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    Blog Post
    ...invariable expression and non-specific, background signal, so you’ll need a range of expression strengths...friendly neighborhood bioinformatician) to look at RNA expression of your gene of interest across a set of human...  From this data, you see that there is high RNA expression in the retina and in parts of the brain, but...that do not express your protein of interest endogenously and transfect them with a plasmid encoding your...protein. Common approaches include using RNAi to knock-down expression of your target or CRISPR/Cas9 to create...Orthogonal Strategies This approach compares target expression of antibody-independent approaches to your antibody-dependent...proteins. Additionally, they require variable expression of your target across samples - it is hard to...

  9. Twenty Years of Sharing: Expanding Accessibility

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    Blog Post
    ...Views Guides CRISPR Guide Lentiviral Guide AAV Guide Optogenetics Guide γ-Retrovirus Guide Protocols... Lentiviral Plasmids Tetracyline Inducible Plasmids Empty Backbones Bacterial Expression Systems AAV ...AAV Plasmids Blogs Origin of Replication What is a Plasmid? How to Design Your gRNA for CRISPR Editing... that made it easy to share plasmids, both for people depositing plasmids and for people requesting them... Verify Your Plasmid Using a Restriction Digest Analysis Videos What is a Plasmid? How to Design...video, however, is “What is a Plasmid?”, An animated explainer of a plasmid and its parts that helps the... was only after creating a plasmid repository and shipping internationally, after all, that we could identify...

  10. Hot Plasmids and Viral Preps - September 2021

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    Blog Post
    ... targets human mRNA binding proteins with 10 guide RNAs per gene. The lab modified the lentiviral backbone...subset of the new plasmids and viral preps 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...of robust expression of high G+C content genes, as validated through monitoring the expression of fluorescent... CRISPR plasmids are always being added to the repository. To find all of the CRISPR plasmids available... Xiao Wang Lab has recently deposited a set of plasmids that helps identify base edited cell populations...populations without end-point sequencing assays. These plasmids use “Cas9-mediated adenosine transient reporter...

  11. The Fluorescent Vegetables in Aptamer Soup

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    Blog Post
    ...processes including RNA movement by tagging RNA sequences with an aptamer and RNA transcription by placing...study of artificial guide RNAs that can be used to simultaneously target cellular RNA to specific intracellular...was developed to quantify RNA transcription by RNA Polymerase III. By expressing Corn under promoters for...named for a fruit or vegetable! What is an RNA aptamer? RNA is not inherently fluorescent and can be challenging...Fluorescent RNA aptamers are used to track cellular processes by transforming cells with plasmids encoding...After 4-6 rounds of SELEX, the RNA pool was cloned into bacterial expression vectors to create a library....fused to a separate piece of RNA. Fluorescence is observed when these RNAs complex and the entire aptamer...

  12. Five Popular Model Organisms

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    Blog Post
    ...knocked-down through RNA interference (RNAi) by feeding the worm bacteria expressing specific plasmids (L4440) that...spatiotemporal expression of a gene of interest. This type of control over gene expression is possible in...signaling in the entire adult brain, using a plasmid that expresses a genetically encoded calcium sensor, GCaMP6...interested in C. elegans plasmids and resources check out Addgene’s Worm Expression page. Looking for more...our quick guide to Drosophila Download the fluorescent proteins 101 eBook Read our plasmids 101 blog series... Browse our plasmid collection pages Read our molecular biology reference Find plasmids for your research... resistant to chemotherapy. You can find these plasmids at Addgene. Another advantage of mice as model...

  13. CRISPR-Cas14: a family of small DNA-targeting enzymes enabling high-fidelity SNP genotyping

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    Blog Post
    ...regions containing the putative tracrRNA into a plasmid and expressed it in an E. coli bacteria strain...on Addgene.org Find the Cas14 plasmids here Checkout our CRISPR guide page Browse the CRISPR collection...cleave ssDNA in trans like Cas13 and Cas12 do for RNA and dsDNA, respectively. However the authors found...activated and can cleave fluorescent probes of dsDNA and RNA in trans. Fluorescent signal indicates the presence...the ultimate combination enabling the detection of RNA, dsDNA, and now ssDNA. By comparing the ability of...certain sequence specificity in the middle of the gRNA to be activated. Cas14 in diagnostics By cutting...SARS-CoV-2) and genetic mutations. By providing a gRNA specific of a viral sequences, for instance, the...

  14. PITChing MMEJ as an Alternative Route for Gene Editing

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    Blog Post
    ... Our CRISPR Guide Pages Find CRISPR Plasmids for Your Experiments Deposit Your New Plasmid Tools with ...cotransfected with a plasmid carrying Cas9, the PITCh-gRNA, and the locus-specifc gRNA. The PITCh-gRNA creates two...The PITCh plasmid contains a GFP-Puro cassette flanked by 5' and 3' microhomology and PITCh-gRNA binding...DSBs in the PITCh plasmid to release the cassette, and an insertion-locus-specific gRNA cleaves genomic ...genome. Open questions and alternative systems The ready-made PITCh plasmids available from Addgene are...Integration into Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about... knock-ins, some labs have used NHEJ for whole plasmid integration; however, since NHEJ is error-prone...

  15. History of CRISPR Cas - A tale of survival and evolution

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    Blog Post
    ... Cas proteins then express and process the CRISPR loci to generate RNA, which guide a Cas nuclease to ...CRISPR/Cas, check out Addgene’s CRISPR Guide or find CRISPR/Cas plasmids for your research. Do you have a cool...that neutralize CRISPR/Cas. Earlier this year, expression of 1 of 5 anti-CRISPR genes were found to inactivate... in the field, and enabling access to multiple plasmids used for various platform applications. For more...

  16. How to Design Your gRNA for CRISPR Genome Editing

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    Blog Post
    ...on Your CRISPR Basics with Our CRISPR Guide Find CRISPR Plasmids for Your Research Find CRISPR Pooled ...of gene expression. Location and sequence are important considerations for designing your gRNAs. For indels...scoring system for selecting a gRNA, as the method of producing the guide (synthetic, in vitro transcription...CRISPRa and CRISPRi Finally, for modulating gene expression at the level of transcription – CRISPRa (activation...ever perfect, but this can decrease the number of guides one needs to test in the lab. Importantly, for ...explanation of our CRISPR resources. He really likes small RNAs.     References Anzalone AV, Koblan LW, Liu DR (...Doench JG (2017) Am I ready for CRISPR? A user’s guide to genetic screens. Nat Rev Genet 19:67–80 . https...

  17. Viral Vectors 101: Transductions

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    Blog Post
    ...HEK-293 cells. When all of these viral plasmids are expressed within the production cell line, viral particles...integrated into the host genome? Expressed episomally long-term? Expressed short-term? Answering these questions...viral particles are packaged, when the vector is expressed. Once the design and construction steps are complete...typically transfected along with viral packaging plasmids into a viral production cell line, usually HEK...Then the viral particles do the rest! Downstream expression or integration resulting from transduction typically...desired event.   Figure 1: Transduction to expression process. A viral particle enters a host cell,...cell, enters the host nucleus, is transcribed into RNA, and is translated into protein.   Is there an...

  18. Optogenetics + CRISPR, Using Light to Control Genome Editing

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    Blog Post
    ...the target DNA sequence as directed by the guide RNA (gRNA), while the CRY2PHR-activator fusion floats...) Development of Light-Activated CRISPR Using Guide RNAs with Photocleavable Protectors. Angew Chem Int... in Cells and Zebrafish using Light‐Activated Guide RNA. Angew Chem Int Ed 59:8998–9003 . https://doi....cells to induce expression of human IL1RN, the researchers saw an 11-fold increase in mRNA levels after ...dCas9 binding occurs. Bubeck et al. expressed a telomere-targeting gRNA and a dCas9 fusion protein with three... that would use light to turn on and off gene expression while imparting spatiotemporal control, reversibility...transcriptional activator domain (VP64 or p65). Upon expression in the cell, the dCas9-CIB1 fusion binds to the...

  19. Single-cell tracking of lineage and identity with CellTag

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    Blog Post
    ...as single cell RNA-seq (scRNA-seq), now allows scientists to identify the gene expression patterns unique...cell page Find plasmids for reprogramming and differentiation Check out our lentivirus guide Sign up to receive...successfully reprogram. Unfortunately scRNA-seq only captures the gene expression of cells at a single time point... that combines scRNA-seq and lineage tracking to track the dynamics of gene expression and clonal history... making it difficult to investigate how early expression patterns influence reprogramming outcomes. CellTag...single-cell level, thus linking ancestral gene expression patterns with reprogramming outcomes (Biddy et...CellTags were detected over a 10-week period. Their expression decreased over time but they were not silenced...

  20. Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

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    Blog Post
    ...al. "Crystal structure of Cas9 in complex with guide RNA and target DNA." Cell 156.5 (2014): 935-949. PubMed...Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA sequences used for targeting; however... Your gRNA Browse Our CRISPR 101 Series Resources on the Addgene Website Find CRISPR Plasmids Browse...evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR is a ground-breaking... modified for genome editing, activating gene expression, visualizing genomic loci, and much more. Now...compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids are available from Addgene here! Future possibilities... (2015): aad5227. PubMed PMID: 26628643. Find plasmids from this publication at Addgene. 2. Nishimasu...
Showing: 81 - 100 of 278 results