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We narrowed to 165 results for: cas9 expression vector

Showing: 21 - 40 of 165 results

  1. Hot Plasmids and Viral Preps - May 2021

    Type
    Blog Post
    ...different promoters driving expression of Cas9 were used in this study.  The zCas9i cloning kit is a plant ...contains a series of vectors and DNA modules for assembly of CRISPR/Cas9 constructs. zCas9i is a high efficiency...terminals with one vector. AAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby from Liqun Luo’s lab expresses membrane tethered...element containing vectors developed in the Jordane Dimidschsteind lab provide expression selectively in ...interneurons. We now provide ready-to-use AAV vectors expressing GCaMP6f with this promoter. Image calcium...dendritic neurites from axonal neurites with AAV vectors expressing cytoplasmic targeted tdTomato and pre-synaptic...efficiency intron-optimized SpCas9-coding gene. SlugCas9 is a compact Cas9 ortholog from Staphylococcus lugdunensis...

  2. Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins

    Type
    Blog Post
    ... cloned into an entry vector. The 2in1 cloning system contains two entry vectors (pUC57-L1L4 and pUC57... It works with the S. pyogenes Cas9, dCas9-effector fusions, and xCas9, and requires no additional chemicals... of protein coexpression. By introducing a single plasmid containing both GOIs, expression levels are ...episode of Hot Plasmids! Optogenetic control of CRISPR-Cas9 by CASANOVA Article contributed by Cary Valley  ...CRISPR systems including the popular S. pyogenes Cas9 (Rauch et al., 2017, Hynes et al., 2017). However..., they have developed a system to control CRISPR-Cas9 activity using blue light (Bubeck et al., 2018)...., inhibiting function of Acr. This allows CRISPR-Cas9 to function on its target.  They name their most...

  3. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    ...ubiquitous expression of sgRNA(s) and mCherry (vector 1) with neuron-specific expression of dCas9-VPR (Figure...system with ubiquitous expression of sgRNA (top) and neuron-specific expression of dCas9-VPR (bottom). Created...large size of Cas9 activators, which largely precludes delivery by adeno-associated vectors (AAV). This ...et al., 2018). This mouse expresses a modified version of the “Suntag” Cas9 activator that recruits up... (i.e., how many sgRNA expression cassettes will fit). Cre-dependent expression of the SPH activator paired...be found here. Neuron-optimized viral vectors Alternatively, Cas9 activators can be delivered to the CNS...Figure 2), a popular Cas9 activator developed by the Church Lab. Neuron-specific expression is mediated by ...

  4. 15 Hot Plasmids from 2017

    Type
    Blog Post
    ...-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity; mammalian expression vector Rees H, et al. Nature...Bacterial Expression Construct SpCas9 Inhibition (E. coli) Mammalian Expression Construct SpCas9 Inhibition..., a base editor containing high fidelity Cas9 variant HF-Cas9. They subsequently tested the original BE3...-BE3  EQR-Cas9 (NGAG)  Lower off-target activity than BE3  85173  pBK-VRER-BE3  VRER-Cas9 (NGCG)   ...pET42b-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity; protein purification vector  87439  pCMV-HF-BE3...and ribozyme systems for multiplex gRNA expression. Expression of toolkit components can be controlled...gene editing. The bacterial and mammalian expression vectors for these CRISPR inhibitors are listed in...

  5. Overcoming the AAV Size Limitation for CRISPR Delivery

    Type
    Blog Post
    ...one AAV, Cas9 must be made smaller. Previous attempts to “shrink” Cas9 include the use of St1Cas9 (~3.3 ...amino acids in length, Cas9 from Campylobacter jejuni (CjCas9) is the smallest Cas9 ortholog characterized...been limited by problems with Cas9 delivery. Adeno-associated viral vectors (AAV) are commonly used for ...group previously packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging...vehicle for Cas9. However, to allow for more room for regulatory sequences while still fitting Cas9 and its...equally potent Cas9 protein, the Zhang lab took a different approach. They analyzed over 600 Cas9 orthologs...Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes...

  6. DAP arrays for multiplex gene editing

    Type
    Blog Post
    ... found led them back towards Cas9 fused base editors (nCas9-BE), as Cas9 is mechanistically more favored...promoter, which is used to drive the expression of the array. After expression, the individual gRNAs are released...also exhibited reduced Cas9-dependent off-target editing and do not cause higher Cas9-independent off-target...leading the edge of the precision gene editing. CRISPR-Cas9, the key component to enable efficient base editing...organisms and has previously been used for multiplex Cas9-nuclease editing. Typically, a single array encoding...number of gRNAs that can be packaged into viral vectors for potential in vivo applications or multiplex...demonstrated that the tRNA-gRNA array can drive the expression of itself, without using any additional promoter...

  7. What's New in CRISPR - September 2019

    Type
    Blog Post
    ...with GeneWeld vectors GeneWeld from Jeffrey Essner’s lab uses CRISPR/Cas9 and a donor vector to create knock-ins...This time: GeneWeld vectors to create knock-ins CasX Drug inducible CRISPR/Cas9 activation E. coli genome-wide...inducible CRISPR/Cas9 systems for transcriptional activation and genome editing by fusing Cas9 to a mutated..., or transcriptional regulators. In this system, Cas9 generates a double-stranded DNA break at the genomic...cargo (ex: fluorescent protein gene) on the donor vector. Therefore, targeting requires two sgRNAS: one ...site and a universal sgRNA that targets the donor vector sequence. Homology arms that flank the donor plasmid... Read the paper in bioRxiv Find the GeneWeld vector kit CasX, a small Cas protein, is active in E....

  8. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    ...types. They have also fused LSD1 to x-Cas9(3.7). x-Cas9(3.7) is a SpCas9 mutant that exhibits increased PAM.... These two vectors are part of the larger pMVP/pMAGIC cloning system, which contains Cas9 fusions for...pCMV-dCas9-D3A) and Tet-dependent (TetO-dCas9-D3A) constructs. For lentiviral expression, Fuw-dCas9-Dnmt3a...deposited mammalian expression constructs including pcDNA-dCas9-p300 Core and pcDNA3.3-Nm-dCas9-p300 Core, as...as well as pLV-dCas9-p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac...activate transcription. A lentiviral vector with the same modifier, Fuw-dCas9-Tet1CD, is available from Rudolf...described above, dCas9-LSD1 inactivates targeted enhancers. Although the lab used this vector in chick embryos...

  9. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ... purifying Cas9? Check out pET-28b-Cas9-His from Alex Schier's lab, designed for expression and purification... of Yonglong Chen, pCS2-3xFLAG-NLS-SpCas9-NLS is a Cas9 expression plasmid that was used by Chen and colleagues...colleagues, FokI-dCas9 expresses Fok1 nuclease domain fused to catalytically inactive Cas9 DNA-binding domain... existing transposon and vector systems and creating an all-synthetic vector that included only the elements... into a destination vector in a predefined order. Finally, the destination vectors are used with Multisite... eight entry vectors can be used for each Multisite Gateway compatible destination vector for a maximum... to Addgene, including lentiviral and AAV expression vectors for certain variants. Klapoetke et al.,...

  10. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    ...A549 cells stably expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast (52962-LV...variable Cas9-expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal... Figure 1: Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting... Cas9 delivery Overview: Before any editing can begin we need our A549 cells to express Cas9. In these...too many Cas9 integrations per cell, and my stable cell pool had wildly variable Cas9 expression (Figure...Checking Cas9 expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9,... 20% of their Cas9-expressing cells. So, even when Cas9 is efficiently editing, don’t expect a 100% knockout...

  11. Hot Plasmids and Viral Preps - March 2021

    Type
    Blog Post
    ...-baToN, “sender” cells express GFP on the surface and “receiver” cells express an anti-GFP nanobody on...response 1 (TIR1) expressed under a desired promoter with a specific spatiotemporal expression pattern and ...and editing with a single Cas9-VPR in Saccharomyces cerevisiae. To control Cas9 activity and influence DNA...Jacobs-Wagner’s lab produce different levels of dCas9 expression and contain different antibiotic resistance... nanobody cross-reacts with BFP, sender cells expressing surface BFP generated BFP-labeled receiver cells...visualized.  The tool-kit consists of highly useful vectors compatible with Gibson and Saptrap cloning systems...for delivering CRISPRi components by a single AAV vector are now available from Tonia Rex's lab.  vfCRISPR...

  12. Hot Plasmids Spring 2024

    Type
    Blog Post
    ... A cis-splicing vector served as positive control (cis-ctrl). v1, vector 1; v2, vector 2; BD, binding ...SARS-CoV-2 research New viral vector preps of Voltron2 voltage sensors AAV vectors for targeting microglia ... offers vectors for CROPseq-multi using selection by Puromycin, Zeocin®, or nuclear expression of BFP2...voltage sensors By Brian O’Neill New viral vector preps expressing the Voltron2 voltage indicator, created...administering these AAVs with a dilute Cre-expressing vector resulted in a sparsely-labeled field of view...high microglia-specific gene expression when incorporated in an AAV vector (Figure 5). Efficiency and specificity...of miR124T was inserted into the vector to silence transgene expression in neurons and other non-microglia...

  13. Viral Vectors 101: Viral Applications

    Type
    Blog Post
    ...interest, turning expression off permanently. Viral vectors can also be used to express proteins. Viral...Browse Our Viral Vector Pages Learn About Viral Vector Biosafety Deposit Your Viral Vectors ...applications Gene expression control Researchers are always wanting to manipulate gene expression – turn it up...even inducibly depending on the vector and virus. Viral CRISPR-Cas9 tools have been engineered to introduce...interest, and often a selectable marker. This vector can express your construct in target cells transiently... of these expression control mechanisms require a specific modification of the viral vector for your gene...We offer a variety of viral vectors, viral services and even a Viral Vectors eBook to get you started. ...

  14. X-CHIME: Context Dependent Germline Knockout in Immune Cells

    Type
    Blog Post
    ...transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic stem cells with a lentiviral vector containing ...utilizes custom mouse strains and lentiviral expression vectors (Table 1). The four systems Name Function...genes pXPR_219 Violet-excited GFP (vex) Rosa26-Cas9 I-CHIME Inducible knockout of a single gene...Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 L-CHIME Knocks out a single gene in defined... Violet-excited GFP (vex) Lineage-Cre; Rosa26-Cas9 S-CHIME Knocks out pairs of genes sequentially...Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME systems Proof of Concept These...Haining, W. N., & Sharpe, A. H. (2019). A CRISPR-Cas9 delivery system for in vivo screening of genes in...

  15. CRISPR Kinome Libraries Available: Pooled and Individual Plasmid Formats

    Type
    Blog Post
    ...are available in two vector formats: all-in-one (lentiCRISPRv2, expressing both Cas9 and an individual gRNA... gRNA from this single vector) and guide-only (lentiGuide expressing only an gRNA, for use with cells ...cells that already have Cas9). Each library consists of approximately 3,000 gRNAs, as opposed to the 70,000...plasmids will also be useful for imaging or gene expression assays necessitating the screening of individual...activity and minimize off-target effects of CRISPR-Cas9." Nature biotechnology (2016). PubMed PMID: 26780180...Learn to Conduct Genome-wide Screens with CRISPR/Cas9 Get John Doench's Tips on gRNA Design Check Out ...

  16. With Our New Viral Service, We're Taking CRISPR Further!

    Type
    Blog Post
    ... delivery of SpCas9 and Cas9 nickase for genome editing. You can also enhance expression of your gene ...must be used to infect a cell line already expressing Cas9. As John Doench (Broad Institute and member...Like all things in science, getting a high titer Cas9 lentivirus prep takes a bit of time and effort that...easy process. The cloning capacity of lentiviral vectors (~8.5 kb of insert) and the reduced titer associated...providing lentiviral preps of select lentiviral vectors from the repository for you. We perform test infections...infections with all of our viral preps and host our expression data (western blots or fluorescent microscopy...gene of interest with a variety of Cas9 activators available in lentiviral format. Finally, while we can’...

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

    Type
    Blog Post
    ...like Cas9. This is where TnpB offers a clear advantage. At approximately one-third the size of Cas9 or ...Practical Guide: Using pK-TnpB1 and pK-TnpB2 Vectors The vectors pK-TnpB1 and pK-TnpB2 are designed for plant...the digested vector. Step 4: Ligation Ligate the annealed oligos into the digested vector using your ... most well-known CRISPR-associated nucleases are Cas9 and Cas12a, the heavyweights in the genome editing...efficient delivery into cells, particularly via viral vectors. While viral delivery is a highly effective method...amino acids in size. To give a visual analogy: if Cas9 or Cas12a is a soccer ball, TnpB is a baseball —...genome editors to date. We have developed multiple vector versions for TnpB-mediated genome editing, and ...

  18. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

    Type
    Blog Post
    ...multiplexed gRNA expression (Wong et al., 2016), in addition to the combinatorial expression of other genetic...CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12 ...below and pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC...barcode, respectively. Once cloned into the storage vector, the pooled, barcoded gRNA library is now ready...pAWp12 lentiviral vector should retain the diversity in your gRNA library, but this should be verified by NGS...activity and minimize off-target effects of CRISPR-Cas9." Nature biotechnology (2016). PubMed PMID: 26780180...transcriptional activation by an engineered CRISPR-Cas9 complex." Nature (2014). PubMed PMID: 25494202. ...

  19. Important Considerations When Using AAVs

    Type
    Blog Post
    ...Tool for Gene Expression in Mammals A Match Made in Heaven: CRISPR/Cas9 and AAV Viral Vector Elements Additional... have found that expression occurs quickly and results in long and stable expression (up to 9 months in...the onset of gene expression. However, it has yet to be determined what gene expression looks like at 3 ...-associated viral vectors: packaging capacity and the role of rep proteins in vector purity." Human gene...Future? I knew little about adeno-associated Viral Vectors (AAVs) before starting my graduate program at the...in locomotion. We have relied heavily upon AAV vectors to deliver chemogenetic/optogenetic constructs ...Although they have a small packaging capacity, AAV vectors were suitable for our needs for the following reasons...

  20. Addgene's Top 10 Blog Posts of 2015

    Type
    Blog Post
    ...repository use Cas9 to cut DNA, Addgene Science Communications Intern Mary Gearing explains how Cas9 homolog,...Match Made in Heaven: CRISPR/Cas9 and AAV Adeno-associated viral (AAV) vectors are useful for in vivo gene...researchers with many different ways to control gene expression. Get the full scoop from Addgenie A. Max Juchheim... rundown of some of the strains you can use to express your protein of interest. 4. Plasmids 101: Blue-White...Blue-White Screening Getting a lot of empty vector contaminants in your cloning experiments and tired of...Validating Your Genome Edit Once you’ve used CRISPR/Cas9 to make a change to the genome, you need to check...Browse Our CRISPR Resources Learn More about Viral Vectors Browse the Repository  ...
Showing: 21 - 40 of 165 results