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We narrowed to 278 results for: guide rna expression plasmid

Showing: 61 - 80 of 278 results

  1. CRISPR 101: Mammalian Expression Systems and Delivery Methods

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    Blog Post
    ...electroporation. Transient expression of CRISPR components, expression decreases as RNA is degraded within the...donor plasmid or single-stranded DNA oligonucleotide, so you will need to make sure your expression system...as either protein, RNA, or DNA. Finally, once you have identified the best expression system, you can then...included with each expression system as well as suitable applications. Expression System Components ...System Application Mammalian expression vector Promoter driving Cas9 expression can be constitutive or inducible...Addgene.org Browse CRISPR Plasmids for Mammalian Systems Check out our CRISPR Guide Pages View CRISPR Protocols... in vivo viral delivery. Cas9 mRNA and gRNA Plasmids containing gRNA and Cas9 are used in in vitro transcription...

  2. Hot Plasmids and Viral Preps - January 2021

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    Blog Post
    ...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...control expression of their genes of interest. While recombinases are powerful gene expression tools in...changes in a protein.    Cells expressing an RFP reporter plasmid for recombinase activity were exposed...highlights from recent CRISPR plasmids. To find all of the CRISPR plasmids available from Addgene, head...AAV-deliverable ABE.  Find the plasmid here Jesse Zalatan’s lab deposited plasmids for a chemically inducible...active site in Casɸ can both process crRNA and carry out the crRNA-guided DNA cutting, unlike other well-characterized...

  3. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

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    Blog Post
    ... can serve as the target for Cas9’s single guide RNA (sgRNA), provided it meets two conditions:  1) The...CRISPR Plasmids Catch up on your CRISPR background with our CRISPR Guide Find Software Tools for gRNA Design...variety of Cas9 containing plasmids that have been optimized for expression in different species and cell...available sgRNA design programs. Find Validated gRNAs for Your Next CRISPR Experiment Cas9 expression systems... system utilizes an RNA template containing the desired edit adjoined to the sgRNA, a reverse transcriptase...fusion and a modified gRNA that is capable of interacting with an additional RNA-binding transcriptional...great choices. Check out our SpCas9 containing plasmids, optimized for a variety of species.   Specificity...

  4. CRISPR 101: Ribonucleoprotein (RNP) Delivery

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    Blog Post
    ...minimum, two components: the Cas9 protein and a guide RNA (gRNA) targeting your genomic site of interest. This...promoters must be chosen for both Cas9 and gRNA expression       Plasmid DNA may be incorporated into the genome...similar efficiency as compared to plasmid-based expression of Cas9/gRNA and can be used for most of the ... EF1A promoters found in many CRISPR plasmids) are not expressed. Because this method does not require... to express and purify His-tagged Cas9 from E. coli using a plasmid such as pET-28b-Cas9-His from Alex...in crops, delivery of Cas9-gRNA RNPs to plants, as an alternative to plasmid based techniques, has been...tips on gRNA design Resources on Addgene.org Browse all CRISPR plasmids Find validated gRNAs Check out...

  5. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

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    ...CRISPR screening method that makes use of guide RNAs with internal barcodes (iBARs) in their loop region....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...digestion of single molecules in real-time. Find plasmids expressing tTALE-FP Shin et al., Sci. Rep. 2019. https...write about a recent plasmid deposit please sign up here.  Human whole genome sgRNA iBAR library Article...the stop cassette is removed, the effector is expressed. By truncating one of the recombination sites ...calcineurin-dependent response elements (CDREs) and drives gene expression. Because calcineurin is required for virulence...

  6. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

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    Blog Post
    ...with Our CRISPR Guide Find CRISPR Plasmids for Your Research Check out Our List of gRNA Design Tools ...in Jacob Corn’s lab. CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts genomic DNA. The...introduces functional Cas9 protein and the targeting guide RNA into cells by electroporation. Results from our...PMC4383097. 3. Kim, Sojung, et al. "Highly efficient RNA-guided genome editing in human cells via delivery of... relatively modest, requiring expressed Cas9 protein, transcribed sgRNA, and optional donor DNA. Chris...validated editing reagents can be found on our plasmid pages. We encourage any labs performing genome ...research with a simple question: how do Cas9 and sgRNA interact with and dissociate from a DNA target? ...

  7. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

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    Blog Post
    ...construction of plasmid vectors and/or rely on cumbersome in vitro synthesis of single guide RNAs (sgRNAs), are ...index.html Design crRNA/ssODN: Using Ensembl Transcript ID(s) as an input query, pick crRNA (guide RNA sequence...two-part cr:tracrRNA system is much more potent than the in vitro-transcribed single guide RNA (IVT sgRNA... developed a “TAG-IN” bioinformatics tool for guide RNA and donor DNA design, and implemented our optimised...successful delivery of three reagents to cells: a guide RNA, Cas9 protein, and a donor DNA that contains the...on the Addgene Blog Plasmids for endogenous gene tagging in human cells Plasmids 101: Protein Tags Fluorescent...Resources on Addgene.org Browse CRISPR plasmids Check out our CRISPR guide pages Find ready-to-use Cas9 lentivirus...

  8. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

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    Blog Post
    ...relative proportion of guide RNAs (gRNAs) across the population. Recall that guide RNAs are the targeting ... Find CRISPR Plasmids for Your Research Check out Our CRISPR Guide Find Validated gRNAs ...compute coordinates for genetic elements like protein, RNA, and DNA (Table 1). As with GPS in the real world...Proteins (for our example later, histones) ChiRP-seq RNA Hi-C DNA (genome-to-genome interactions) DNase-seq... DNA compaction, an enhancer can increase the expression of a gene that is millions of base pairs away...Putting it all together, we get the following expression for step 1: (H3K4me1 + H3K27ac) – H3K4me3 The...enhancers with CRISPR Armed with the genome math expression for NANOG enhancers, we’re ready for step 2: ...

  9. Hot Plasmids - August 2020

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    Blog Post
    ... ways to modulate the expression of nearly any gene via specific guide RNA (gRNA) sequences. But these...Addgene #140702) The pMAK461-64 plasmids were expressed in SHuffle T7 Express competent E.coli (NEB) using...approaches rely on continual expression of both dCas9 and the gRNA. This expression can vary significantly ... out these plasmids for piggyBac gRNA delivery and the all-in-one dCas9 targeting plasmids! Hazelbaker...editors, gRNA backbones, and promoters for expression in monocots and dicots. Find the plasmids at Addgene...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...

  10. Overcoming the AAV Size Limitation for CRISPR Delivery

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    ...’s packaging limit. For CRISPR-based RNA editing, the REPAIR (RNA Editing for Programmable A to I Replacement...2019) CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566:218–223 . https://...2014) Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell 156:935–949. https://doi.... on Addgene.org Check out our CRISPR Guide Find CRISPR Plasmids ...catalytically dead dCas13b to the catalytic domain of RNA deaminase ADAR2. Constructs containing the ADAR2 ...capabilities of full-length SpCas9. This set of plasmids includes AAV-Cas9C-VPR for targeted gene activation...activation. Split inteins have also been used to express base editors. References Chew WL, Tabebordbar ...

  11. Genome-wide Screening Using CRISPR

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    Blog Post
    ...in a 1-plasmid system, in which Cas9 is included on the gRNA-containing plasmid, or a 2-plasmid system...single stranded guide RNA (gRNA). The ~20 nucleotide targeting sequence within the gRNA is defined by the...contain thousands of gRNA plasmids, discerned only by a unique barcode on each plasmid. As such, sequencing...Addgene: Find CRISPR Plasmids Read our CRISPR Guide Find tools to design your gRNA ...Read our Quick Guide to All Things Lentivirus Get Tips on Titering Your Lentivirus Plasmids 101: NGS Quality...libraries are composed of many different gRNA-containing plasmids, each with a different genomic target....target sequences and ease of generating gRNA-containing plasmids. CRISPR genome wide-screening experiments...

  12. Plasmids 101: Using Transposons in the Lab

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    ...move antibiotic resistance genes between plasmids or from plasmids to the bacteria’s genome. In humans, if...regions, gene disruption is less of a concern. RNA guided transposons insertion One drawback to DNA transposons...INTEGRATE (Insertion of Transposable Elements by Guide RNA-Assisted Targeting) system. INTEGRATE is based...bacteria.   Figure 3: INTEGRATE system for RNA guided transposon insertion in bacterial genomes. A)...Components of the INTEGRATE system. B) Mechanisms of RNA-guided transposition with INTEGRATE. The QCascade complex...transgenic animals, gene transfer). The discovery of RNA guided transposons in bacteria sets the stage for combining...system has four major components: 1) a CRISPR RNA (crRNA), 2) four proteins (TniQ, Cas8, Cas7, Cas6) that...

  13. New CRISPR Tools: Cas7-11 and PASTE

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    Blog Post
    ...and a guide RNA. The enzyme has dual activities: processing pre-CRISPR RNA into mature CRISPR RNA and cleaving...inserted it into E. coli to express and then test for any proteins that could cut RNA or DNA via a CRISPR mechanism...cuts on the RNA target. In addition, when they looked at the transformed E. coli expressing Cas7-11, they...development of low-toxicity RNA editing tools.   Fig. 1 Schematic of luciferase RNA knockdown assays using...CRISPR 101: RNA Editing With Cas13  CRISPR Cheat Sheet Resources at Addgene.org CRISPR Guide...larger than Cas9, can also be used with mRNA and a synthetic guide. Like PRIME, PASTE relies on single-stranded...they named Cas7-11. Testing showed that Cas7-11 cut RNA with unprecedented specificity: when purified, it...

  14. Viral Vectors 101: The Retroviral Lifecycle

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    ... Addgene's Lentiviral Guide Most popular lentiviral plasmids γ-Retrovirus Guide References Cullen, B....instead get expression of the sensor, marker, or whatever other tool you chose to express. In the case...1973). RNA-directed DNA polymerase of Rous sarcoma virus: Initiation of synthesis with 70 S viral RNA as ...Central Dogma of Biology (gDNA → mRNA → protein): their roadmap includes RNA → DNA (Cooper & Temin, 1974),...cells (i.e. transferring DNA), yet retroviruses are RNA-containing viruses  (Robinson et al., 1965)! You ...that the virus must have its structural proteins expressed from a reverse-transcribed piece of DNA called... follows the release of the two strands of viral RNA and of the viral RT protein (Faras et al., 1973)....

  15. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

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    Blog Post
    ...safe-targeting guides vs. non-targeting guides on screen analysis and hit calling. 5,644 non-targeting guides and...effects of guide expression and dsDNA breaks. Figure 2: Non-targeting vs. safe-targeting guides. The...short guides had reduced toxicity compared to full-length guides. Reduced toxicity of short guides could...non-targeting guides an appropriate control? Many CRISPR screens use non-targeting guides as negative controls...screens. Do short guides (17-18 bp) have less off-target cutting than full-length guides (19-20bp)?  Small-scale...predicted off-target activity when selecting guides. Guides were delivered as a pooled library via a lentiviral...Safe targeting guides better control for non-specific toxicity than non-targeting guides First, Morgens...

  16. Hot Plasmids - February 2022

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    Blog Post
    ...Cas 9 vs. Cas 12a expression cassettes when encoding multiple guides. While two guides require two arrays...individually expressed proteins run in separate lanes. B: individually expressed and coexpressed proteins ...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...editing within a single expression cassette is advantageous to Cas9’s larger expression requirements (Figure...Image from Santilli, et al. 2021 Plasmids are used to express 10, 15, 20, 30, 40, 50, 60, 80 and 100... simple, inexpensive plasmid-based ladder system is a cost-effective alternative to commercial ladders...

  17. CRISPR 101: Cas9 vs. The Other Cas(s)

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    .... Cas13 Cas13 is an RNA-guided nuclease, however, unlike Cas9, it targets RNA instead of DNA. This makes...powerhouse, and for good reason. It is guided by an RNA molecule, the gRNA, to a complementary site in the genome...indiscriminately cleaves ssRNA when engaged at its RNA target sequence. The RNA-targeting ability of Cas13... cleave RNA through the same mechanism as traditional spCas9. If you are looking to modify RNA, Cas9 just...degrade target viral RNA. For more information on Cas13, check out our blog on RNA editing. Fast facts...proposed to be useful for the same applications (RNA knockdown, RNA editing, etc.). The main differences between...non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral RNA cleavage and ...

  18. Plasmids 101: Inducible Promoters

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    ...Resources on the Addgene Blog: Plasmids 101: The Promoter Region Plasmids 101: Cre-lox Light-Switchable...Tet) Inducible Systems at Addgene Cre-lox Guide and Plasmids     ...Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region ...gene, it therefore determines the timing of gene expression and largely defines the amount of recombinant...lac inducible system is found in many bacterial expression vectors. Negative inducible promoter pBad is ...Promoter Subtype Promoter Example Activator Addgene Plasmid/Kit Alcohol-regulated AlcA promoter AlcR pGGA008...Temperature inducible promoters Temperature sensitive expression systems are typically less leaky than chemically...

  19. Tips for Titering Your Lentiviral Preps

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    Blog Post
    ...your virus and assaying for expression of a gene carried on the transfer plasmid or quantifying the number...particles. Direct measurement of lentiviral RNA is one alternative to direct p24 measurement. Like a p24 ELISA...direct p24 measurements via ELISA and qPCR for viral RNA. Measuring p24 levels in a viral prep is straightforward...expensive than an ELISA kit. In this approach, viral RNA is first converted to cDNA and then quantified using...beneficial as they ensure that the correct transfer plasmid was used in the transfection; if preparing several...the p24 assay, titering via measurement of viral RNA can overestimate the amount of infectious virus in... that comes from amplification of the original plasmid which can over-inflate results. A packing vector...

  20. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

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    ...CRISPR interference plasmids Find bacterial CRISPR plasmids Read our CRISPR guide ...transposon system. This system has two parts: a plasmid expressing the Tn7 transposition genes (tnsA, tnsB, ... human gut flora. Guides targeted essential and non-essential genes with two guides per gene. The library... CRISPRi knockdown. As expected, bacteria expressing guides that target essential genes were less represented..., tnsC, tnsD, and tnsE), and a plasmid that contains the Mobile CRISPRi cassette flanked by left and right...copy of the RP4 transfer machinery mobilize these plasmids for transfer to recipient cells during a tri-parental...Mobile-CRISPRi ICE element forms a conjugative plasmid-like DNA circle and is transferred to recipient...
Showing: 61 - 80 of 278 results