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We narrowed to 113 results for: dcas9

Showing: 1 - 20 of 113 results

  1. Simple CRISPR-based Epigenetic Editing: dCas9-directed DNA Demethylation

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    Blog Post
    ...Enzymatic domains tethered to dCas9 often interact with DNA independently of dCas9 targeting (Galonska 2018...binding is crucial for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable...has several great options: pLV hUbC-dCas9-T2A-GFP for removable dCas9 expression and pLM-CMV-R-Cre. Selection... Some enzymatic fusions are godsends. Tethering dCas9 to the histone acetyltransferase p300 or the DNA...absent before. But other enzymatic fusions, such as dCas9-TET, can, like the iron ball-and-chain that they...-based epigenetic approach (spoiler alert: it’s dCas9… with no modifications). Ready to get started?   ...dioxygenase  When I started to work on building a CRISPR/dCas9-based tool for DNA demethylation in 2015, none existed...

  2. CRISPR 101: Epigenetics and Editing the Epigenome

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    Blog Post
    ...including pcDNA-dCas9-p300 Core and pcDNA3.3-Nm-dCas9-p300 Core, as well as pLV-dCas9-p300-P2A-PuroR for...pCMV-dCas9-D3A) and Tet-dependent (TetO-dCas9-D3A) constructs. For lentiviral expression, Fuw-dCas9-Dnmt3a...Goodell's lab has deposited pcDNA3.1-dCas9-MQ1(Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase...Fusing dCas9 to a transcriptional activator like VP64 or VPR activates transcription, whereas dCas9-KRAB...activators/repressors - dCas9-p300 increased transcriptional activation more than dCas9-VP64, especially when...Non-editing CRISPR applications direct catalytically dead dCas9 fused to a variety of epigenetic modifiers to specific...Transcriptional activation p300 acetyltransferase dCas9 fused to the catalytic domain of p300 acetyltransferase...

  3. Isolating Genomic Regions of Interest with the CRISPR System

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    Blog Post
    ...inactive form of Cas9 known as dCas9 (Jinek et al., 2012). Like Cas9, dCas9 can bind to a specific DNA sequence...targeting gRNA. But dCas9 does not cleave the DNA. Much of the research using dCas9 has focused on transcriptional...mammalian cells by expressing dCas9 with an N-terminal 3xFLAG tag (3xFLAG-dCas9) and a gRNA targeting their...activators (Qi et al., 2013). However, the fusion of dCas9 with a protein tag allows for the isolation of a...genomic region using the CRISPR system, consisting of dCas9 and targeting gRNA, to identify molecular interactions...3xFLAG tag, allowed these researchers to isolate dCas9 with the bound genomic locus, which they then confirmed..., 2009). Other forms of enChIP, that do not use dCas9, require Zinc-finger or TAL proteins targeting a...

  4. CRISPR Activators: A Comparison Between dCas9-VP64, SAM, SunTag, VPR, and More!

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    ... cell lines.   dCas9-VP64 Description CRISPR activation can occur through fusing dCas9 with VP64, a strong... for CRISPR-based gene activation. dCas9-CBP In this method, dCas9 is fused to CBP, an histone acetyltransferase... the programmable guide RNA. Gene activation by dCas9, also referred to as CRISPRa, was initially published...methods for CRISPR activation CRISPR activation uses dCas9, a CRISPR protein variant lacking its endonuclease...shown significant improvements upon the initial dCas9-VP64 method, so there are multiple options to choose...strong transcriptional activation domain. Guided by dCas9, VP64 recruits transcriptional machinery to specific...depending on which sequence is targeted. Performance dCas9-VP64 activation is generally thought of as the “...

  5. What's New in CRISPR - March 2020

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    Blog Post
    ...fluorescent dCas9-KRAB and dCas9-VPR for quantifying and tracking cells that express both the dCas9 construct...Saccharomyces cerevisiae. The system consists of dCas9, the mammalian transcriptional repressor Mxi1, and...accessible and the complex enters the nucleus where dCas9-Mxi1 can repress gene expression. In the dark, the...Caulobacter cells for ctrA gene expression using various dCas9. Image adapted from Guzzo et al., 2020. Caulobacter...Caulobacter genes using a catalytically dead Cas9 (dCas9) derived from Streptococcus pasteurianus  or the...essential cell cycle regulator. The lab expressed both dCas9 and the ctrA sgRNA and saw that the levels of ctrA...minutes in xylose (which controls expression of dCas9). The lab demonstrated the system can also be used...

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

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    ...genes using dCas9-based activators The simplest of the dCas9 activators consists of dCas9 fused to a single...Repressing target genes using dCas9-based repressors Early experiments using dCas9 in bacteria demonstrated... Fig. 3: Cas9 repressors include dCas9 alone and a dCas9-KRAB fusion, both targeted to the promoter...even making a break.  Base editors are dead Cas9 (dCas9) or nCas9 enzymes fused to either cytosine or adenosine...genome but still want to affect gene expression? dCas9 can be used as a platform to deliver various cargo...specific DNA locus by directly fusing the cargo to dCas9.  This technique has been exploited to localize ...demonstrated that targeting dCas9 to transcriptional start sites was sufficient to repress transcription by blocking...

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

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    Blog Post
    .... Using dCas9 as an on/off switch The multiple scRNAs all individually utilize the same dCas9 protein....modulate dCas9 expression, multiple scRNA transcriptional programs can be regulated with dCas9 as a master...an orthogonal dCas9 protein that will still recognize guide RNA but with a different dCas9 binding module... consists of a catalytically dead Cas9 protein (dCas9) that lacks endonuclease activity coupled with a...specific proteins via aptamer domains. Combined with dCas9, this new scaffold RNA (scRNA) will bind to a PAM-adjacent...layered with distinct programs controlled by each dCas9 regulatory switch. By no means limited to the field...applied to effectively change metabolic flux. The dCas9 protein and orthogonal variants can be used as regulatory...

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

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    Blog Post
    ...in which dCas9 binding occurs. Bubeck et al. expressed a telomere-targeting gRNA and a dCas9 fusion protein.... sphaeroides Cas9, dCas9 Gene editing Zhou et al., 2018   pdDronpa Cas9, dCas9 Gene editing, transcription...Shining light on transcriptional activation using dCas9 Initial photoactivatable CRISPR systems published...genomic anchor - an inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 ...(VP64 or p65). Upon expression in the cell, the dCas9-CIB1 fusion binds to the target DNA sequence as ...proteins. The best performing combination was NLS-dCas9-trCIB1 and NLSx3-CRYPHR-p65 - it had the lowest ...The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment of CIB1...

  9. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

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    Blog Post
    ...co-expressed, the gRNA targets the dCas9 to the loci of interest and dCas9 is biotinylated by BirA. From here...To increase the resolution, the Xu lab created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ... cell line that co-expresses three components: dCas9 with an added biotin acceptor site BirA, a biotin...be pulled down via streptavidin IP. Biotinylated dCas9 goes along for the ride throughout all of these ... CAPTURE cell lines will have varying levels of dCas9 and gRNA expression. Additionally, CAPTURE 3C-based...CAPTURE 2.0:  Replace the biotinalatable tag on dCas9 with a BioTAP-tag. The BioTAP-tag is a 69 aa long...three to two. Lentiviral delivery of BioTAP-tagged dCas9 and gRNAs. Using lentiviral delivery of the two ...

  10. CRISPRainbow and Genome Visualization

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    Blog Post
    ...bright-field image. Image from Ma et al, 2016. Using dCas9 to visualize genomic loci in vivo The flexibility... and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing, but it retains its ...By using a gRNA to target fluorescently labeled dCas9 to a given genomic locus, you can track the localization...to noise ratio of the system, they modified GFP-dCas9 and the gRNA scaffold to enhance complex assembly...amount of background fluorescence from unbound GFP-dCas9. With these improvements, labeling efficiency was...different PAM sequence, a gRNA designed for one dCas9 should be specific to that ortholog and not cross-talk...with the other orthologs. Ma et al. tested their dCas9 variants using gRNAs specific for telomeric sequences...

  11. Hot Plasmids - May 2022

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    Blog Post
    ...inactive dCas9 and microbial SSAPs (dCas9-SSAP) which insert genes in a cleavage free manner. The dCas9-SSAP...cleavage-free dCas9-based gene editor using microbial SSAPs. Image from Wang, et al, 2022.  Find dCas9-SSAP...Cre-mediated AAV Targeted Evolution method (M-CREATE) dCas9-SSAP with reduced error rate for long sequence knock-ins... https://doi.org/10.1038/s41593-021-00969-4.   dCas9-SSAP with reduced error rate for long sequence knock-ins...Wilson Le Cong’s lab has developed a method using dCas9-SSAP to knock-in long sequences with reduced risks...could potentially be packaged into an AAV system. dCas9-SSAP will make the editing of kilobase-scaled sequences...

  12. 28 Hot Plasmid Technologies from 2015

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    Blog Post
    ...specific genes using dCas9-VP64 activator fragments (dCas9(C)-FKBP-2xNLS-VP64 and dCas9(N)-FRB-NES). This...gene activation when fused to dCas9. In the dCas9-SunTag-VP64 system, dCas9 is fused to a scaffold containing...domains onto dCas9 to generate a dCas9-VP64-p65-Rta fusion protein (referred to as dCas9-VPR) had a synergistic...transactivating capabilities of CRISPR/dCas9-fusion proteins and have made their dCas9 transactivators available through... through Addgene. The development of dCas9 and dCas9-fusion proteins has dramatically increased the potential...greater than the original dCas9-VP64 activator. They also revealed that dCas9-VPR mediated gene activation...proven challenging using the existing dCas9-VP64 activator. The dCas9-VPR activation plasmids have great ...

  13. Mapping the 4D nucleome with CRISPR/Cas9

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    Blog Post
    ... organization may be altered in disease states. dCas9 gets a colorful makeover Even without its nuclease...nuclease activity, catalytically dead Cas9 (dCas9) has many applications, the most well-known being transcriptional...transcriptional activation/repression. dCas9 has also previously been adapted to fluorescently label a sequence...required by each ortholog, a gRNA designed for one dCas9 should be specific to that ortholog and not cross-talk...with the other orthologs. Ma et al. tested their dCas9 variants using gRNAs specific for telomeric sequences...and showed that different fluorescently labeled dCas9 isoforms are efficiently directed to the proper ...

  14. Hot Plasmids - August 2020

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    Blog Post
    ...an EGFP reporter. Expression of the dCas9-KRAB activator or dCas9-VPR repressor in hPSCs is also driven...piggyBac gRNA delivery and the all-in-one dCas9-KRAB and dCas9-VPR targeting plasmids can be found at Addgene...approaches rely on continual expression of both dCas9 and the gRNA. This expression can vary significantly...expression of single or multiplexed gRNAs and of dCas9 fusions. The dCas-KRAB activator and the dCas-VPR... could quantify and track cells expressing both dCas9 and gRNAs. The multi-gRNA piggyBac vector ...plasmids for piggyBac gRNA delivery and the all-in-one dCas9 targeting plasmids! Hazelbaker et al., Sci Reports...

  15. New Tools Enable CRISPRa for Neuroscience Applications

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    Blog Post
    ..., CRISPRa uses a nuclease-dead variant of Cas9 (dCas9) and engineered combinations of transcriptional ... transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct). More...mCherry (vector 1) with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed...of sgRNA (top) and neuron-specific expression of dCas9-VPR (bottom). Created with BioRender.com. The...investigation. Co-transduction of primary rat neurons with dCas9-VPR and sgRNAs targeting either transcript variant...coding sequence. ES cells that stably express the dCas9-Suntag activator were transduced with a pooled sgRNA...Katherine E., et al. "A neuron-optimized CRISPR/dCas9 activation system for robust and specific gene regulation...

  16. What's New in CRISPR - May 2019

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    Blog Post
    ...After testing dCas9 fusions with transcriptional repressors, they found that fusing dCas9 to an Mxi1 repressor...gene. Read the paper in mSphere Find the dCas9 and dCas9-Mxi1 plasmids If you have a new CRISPR tool...

  17. New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

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    ...an inactive RNA-guided DNA binding protein (dCas9). dCas9 is then tethered to a repressive domain, such...loss of function while nuclease-deactivated Cas9 (dCas9) can be tethered to a repressor (CRISPRi) or an ...library In addition to inhibiting gene expression, dCas9 can also be used for transcriptional activation....developed to recruit transcriptional machinery to dCas9 and its RNA directed binding site. These methods... fusion of an activation domain such as VP16 to dCas9 or the recruitment of multiple copies of VP16 termed... with only two constructs (the other containing dCas9-VP16). Like CRISPRi, CRISPRa is only effective when...

  18. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

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    Blog Post
    ...identical dissociation rates for catalytically inactive dCas9 molecules. We followed these experiments with more...duplex. Image from Richardson et al., 2016.   dCas9 enables sequence replacement without cutting Our...results also demonstrated that catalytically inactive dCas9 produced a “bubble” structure in uncut target DNA... sequence replacement in cells. Targeting three dCas9 molecules to a precisely spaced region allowed sequence...cutting. We still don’t know the mechanism underlying dCas9 editing, but it could be very useful to tackle genetic...

  19. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

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    Blog Post
    ...targeting either a catalytically dead Cas9 (dCas9) alone or a dCas9 fused to the omega subunit of RNA polymerase...tuberculosis. dCas9 and a gRNA are expressed on pRH2502 and pRH2521, respectively. Both dCas9 and the gRNA...2013). As in other systems, catalytically dead (dCas9) targeted to a promoter or gene body can repress... in Gram-negatives) but share the common CRISPR-dCas9 system for targeted repression of gene expression...robust tool for CRISPRi and CRISPRa using RNAP-Omega-dCas9 fusions. Here’s some others. SoxS based CRISPRa ...non-model bacteria. The authors were able to engineer a dCas9 variant fused to bacterial enhancer binding protein...enzyme capable of converting one base to another to dCas9. The typical gRNA targeting approach is then used...

  20. 8 Great Depositor Research Stories

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    Blog Post
    ...Sapozhnikov tells us how to use dCas9 (with no modifications!) as a CRISPR/dCas9-based tool for DNA demethylation...ancestral bias in CRISPR guide library design.  dCas9-directed DNA Demethylation: Guest blogger Daniel...
Showing: 1 - 20 of 113 results