We narrowed to 84 results for: cas9 expression construct

...protein can then be co-expressed in essentially any transgenic zebrafish line expressing a GFP-tagged version...display increased neuronal expression in the brain while having decreased expression in the liver after systemic...only AAV.CAP-B10 causes brain wide expression, with no liver expression, after retro-orbital sinus vein ...Increased neuronal expression using Cre-mediated AAV Targeted Evolution method (M-CREATE) dCas9-SSAP with reduced...10.1038/s41593-021-00969-4.   Increased neuronal expression using Cre-mediated AAV Targeted Evolution method...vein injection of a CAG-driven mNeonGreen construct in mouse. Image from Goertsen D, et al., 2022.  Find...of up to 20%, comparable to commonly used CRISPR/Cas9 methods for large inserts. However, this system ...

...to edit a gene, the expression of Cas9 is no longer necessary. However, Cas9 can be toxic if continuously...leading to transient expression of gene products (Wu et al., 2004). Over time, the expression diminishes as ...2008). 2. Transient expression of transgenes/shRNAs If you want your cells to express a protein or nucleic...the expression of the delivered payload is gradually reduced. For example, after using CRISPR/Cas9 to ...continuously expressed (Fu et al., 2013). Using IDLVs ensures transient expression without the risks associated...associated with prolonged expression. 3. Stable expression in non-dividing cells IDLVs can also be useful for...like creating stable cell lines, delivering CRISPR/Cas9 to cells for genome editing, and introducing inducible...

...lab, if you read a paper and saw an interesting construct you wished to use, you could either make it yourself...Addgene makes it so much easier to share your constructs from lab to lab. Promega supports the Addgene...endogenous tagged genes generated through CRISPR/Cas9 knock-in. Finally, NLuc is very inviting for endogenous...only 19 kDa. An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined...fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged into a retrovirus...reporter was used to sort successfully infected/expressing cells by flow cytometry. The isolated cells were...reporter can improve deep tissue imaging. The constructs for GpNLuc and OgNLuc are available through Addgene...

...Blainey’s lab has developed a multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi...using selection by Puromycin, Zeocin®, or nuclear expression of BFP2.     Figure 1: CROPseq-multi ...produced 90% accurate integration of multiplexed constructs into target genomes and a 10-fold improvement...Anzalone et al., 2019, including modifications to the Cas9 or reverse transcriptase enzymes, inhibition of ...end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed mechanism for engineered...sensors By Brian O’Neill New viral vector preps expressing the Voltron2 voltage indicator, created by the...that administering these AAVs with a dilute Cre-expressing vector resulted in a sparsely-labeled field of...

...involves the expression of long hairpin RNA (hpRNA). This method is facilitated by DNA constructs that enable...research and many different constructs are available to the community. Each construct has unique features, such...transgene. Typically, the desired target of RNAi is expressed as inverted repeats separated by a loop region... such as inducible expression of the hpRNA and/or fusing your gene target sequence to a fluorescent reporter...sequence (discussed below). Examples of these constructs include pGAPi, which allows easy creation of ...reporter. This is achieved at the level of the DNA construct, where your RNAi-target is fused to the DNA backbone...phenotypes quantified. Fluorescent reporter-based RNAi constructs have been designed and widely implemented in ...

...co-conversion markers. Expanding beyond Cas9 Since the development of CRISPR/Cas9 editing methods, other Cas proteins...and yeast cells using modified Cas9 proteins to regulate gene expression (CRISPRi/CRISPRa), visualize specific...University of California, Santa Cruz. Emerging CRISPR/Cas9 editing technologies have transformed the palette...regulation during developmental processes, CRISPR/Cas9 allows us to delete sequences and introduce mutations...remain in the edited animal. Development of CRISPR/Cas9 editing strategies in C. elegans As brilliantly ...step as simple as heat-shocking to induce the expression of a Cre recombinase contained within the knock-in...reported in 2017 uses the direct injection of a CRISPR/Cas9 ribonucleoprotein complex with linear DNA as repair...

...biotin ligases Expression system Stable cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and ...components are co-expressed, the gRNA targets the dCas9 to the loci of interest and dCas9 is biotinylated...creation of a stable cell line that co-expresses three components: dCas9 with an added biotin acceptor site...cell lines will have varying levels of dCas9 and gRNA expression. Additionally, CAPTURE 3C-based sequencing...that is recognized by biotin ligases normally expressed in eukaryotic cells. Using this tag reduces the...  A second application for CAPTURE 2.0 is the construction of spatial and hierarchical chromatin interaction...conformation data is compared with changes in gene expression, chromatin accessibility, and epigenetic modifications...

...plasmids, catalytically inactive Cas9 (dSpyCas9) fused to APEX2 is expressed from a Tet/Shield1 inducible ...Yale University recently deposited a plasmid for expressing a novel reverse transcriptase (RT), MarathonRT...properties make nanobodies easier to transform and express in bacteria, which, in turn, makes them more adaptable...with biotin as an affinity tag. This ‘Caspex’ construct is guided by a pre-defined sgRNA sequence to a...targeted to a sequence of interest with an sgRNA expressed from a separate plasmid. When cells are treated...genomic loci. He made use of catalytically inactive dCas9 by fusing it to an engineered peroxidase, (APEX)...are enriched in the 20 nm radius surrounding the dSpyCas9-APEX2 fusion.  This Sontheimer lab used this technology...

...Tight control of toxin expression is absolutely necessary, because leaky expression of toxin could select...introduce a mutation in the chromosome is to disrupt expression of a gene with an antibiotic resistance cassette...in the chromosome, sometimes interfering with expression of surrounding genes. However, there are ways...(Thomason et al., 2014). In recent years, CRISPR-Cas9 technology has also aided in the creation of scarless... the second crossover step, they tested their expression under both rhamnose- and arabinose-inducible ...  Giacalone, Matthew J., et al. "Toxic protein expression in Escherichia coli using a rhamnose-based tightly...Valentovich LN. "DNA tuner: a computer program for the construction of polylinker sequences of molecular vectors...

...modulate dCas9 expression, multiple scRNA transcriptional programs can be regulated with dCas9 as a master...) model consists of a catalytically dead Cas9 protein (dCas9) that lacks endonuclease activity coupled...gene expression. This strategy can be applied to effectively change metabolic flux. The dCas9 protein...recruit a transcriptional regulator to modulate expression at that locus. The use of regulators allows a... a greater degree of control of desired expression levels dependent upon the type of transcriptional regulator...are comparatively small allowing multiple sgRNA constructs to be introduced simultaneously in a single plasmid...various transcriptional regulators to alter gene expression at the targeted site. Multiple scaffold RNAs...

... third of the size of Cas9). Figure 2: Comparison of TnpB (A) and Cas9 (B) genome editors. Effector...team characterized and deposited a variety of constructs for visualizing cellular components (nucleus,... mGold2. Top: TIRF-SIM imaging of COS-7 cells expressing LifeAct fused to mVenus, mGold2s, or mGold2t...., recruited by either the ωRNA (TnpB) or a gRNA (Cas9). Drawings not to scale. Created with BioRender.... synapses. In addition, ATLAS itself could be expressed Cre-dependently to allow tracing exclusively from...

... Figure 2: (A) Streamlined VchINT expression constructs. (B) High integration efficiencies achieved...effectors employed by Type II systems (such as CRISPR-Cas9). Unlike in regular Type I CRISPR-Cas systems, however...separate plasmids and multiple promoters were used to express the necessary components in E. coli (Klompe et ...). Recently, the lab introduced a streamlined expression approach (Fig. 2A), involving two-plasmid or ...two-plasmid system, an effector plasmid (pEffector) expresses all seven required genes and the CRISPR RNA under... >99% on-target specificity. Additionally, by expressing multiple crRNAs in parallel (Fig. 3), multiplexed...bacteria, alongside more established tools such as Cas9-recombineering, CRISPRa/i, and base editing. Leveraging...

...has a low quantum yield, and when transiently expressed in cells, generates much less signal than comparable...permit five-color fluorescent imaging! These eNL constructs use the brightest characterized luciferase, NanoLuc...photobleaching and phototoxicity are a concern, these constructs come in very handy. This approach is especially... endogenous protein fusions created using CRISPR/Cas9. eNLs offer high luminescent signal for imaging ...

...interest. To get the molecular components of CRISPR/Cas9 (Cas9 protein and the crRNA-tracrRNA complex) and the...regulatory control. But even with the advent of CRISPR/Cas9 technology, inserting large tags into a precise ...We’ve created and tested plasmids that use CRISPR/Cas9 to endogenously tag a wide variety of genes with...guidance on how to make your own gene-tagging constructs below. Read on to find out how these plasmids...process following the double strand break CRISPR/Cas9 makes at the target site. The whole segment—a GFP...the plasmid from being cut and destroyed by CRISPR/Cas9 during transfection. Making the double strand break...the fluorescent tag to the cells, we used CRISPR/Cas9 to make a precise cut in the genome near either ...

...have deposited the best performing sensors and constructs encoding corresponding portions of the Zika virus...virus research community. Find the Zika Sensor Constructs at Addgene As we report, we have embedded this...amplification, new toehold switch designs, CRISPR/Cas9-mediated genotyping, as well as our electronic reader...switches: de-novo-designed regulators of gene expression." Cell 159.4 (2014): 925-939. PubMed PMID: 25417166...

... 2 (Th2) cells from mice that constitutively express Cas9 and screen for genes that affect Th2 differentiation...differentiation and activation, using expression of differentiation factors as the screen readout. This retroviral... voltage changes in worms and have made these constructs available at Addgene. These include Arch(D95N...Wang Lab created a tripartite system consisting of dCas9, a gRNA fused to an RNA binding motif, and a RNA-binding...protein-bEFP fusion. Thus, the bEFPs bind the UAS via the dCas9-gRNA complex which allows transcription from the...

...to create transgenic mice expressing fluorescent protein reporter constructs in the immune lineage. By...loxP sites such that their expression is turned on or off in cells expressing Cre. The result of a system...allows the visualization of fluorescent proteins expressed in the specimen.  2. Fluorescence Activated Cell...immune lineage, scientists could easily track GFP expressing cells. For example, by harvesting lymph nodes...above depicts the infiltration of neutrophils expressing LysM-GFP (green) into a draining lymph node after... efficiently in two-photon microscopy and new constructs have facilitated the creation of mouse lines ...other technologies such as optogenetics and CRISPR/Cas9 will help immunologists create even better tools...

..., a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3 showed 37-fold...other methods. In a head-to-head comparison with Cas9, Cas9 modified 9/12 known off-targets with a 14% indel...individual Cas9. They also mutagenized the cytidine deaminase portion of the base editor to create SpCas9 base...et al., 2020 SpRY Cas9, available fused to ABEmax(7.10) or to BE4max   Evolved SpCas9 with NRN or NYN PAM... adenine base editors by expression optimization and ancestral reconstruction. Nature Biotechnology, 36... 1.1%. These numbers are a vast improvement over Cas9-mediated HDR for the loci tested, where average ...system is also subject to off-target editing based on Cas9 off-target activity.  Find CRISPR base editing plasmids...

... of expression. For example, CAG is a strong promoter that drives very high, ubiquitous expression, while...knowledge to be able to design a construct for the successful and specific expression of your gene of interest...such as SaCas9, CRISPR gRNAs, or your gene of interest. Promoter The promoter drives the expression of the... prevent ITR loss, it’s best to propagate AAV constructs in recombination-deficient bacterial strains,...type-specific, which can help you restrict the expression of your transgene to a specific species or location...to spatially and temporally control transgene expression. Lox sites are directional 34 bp sequences that...nuclear export of mRNA and helps increase the expression of your transgene. The WPRE is located at the...

...to fertility. Several zebrafish-specific gene expression databases that provide information about when...have been developed to help parse and visualize expression data in useful ways. Zebrafish mutants In the...zinc-finger nucleases, TALENs, and more recently CRISPR/Cas9. Another popular targeted gene deactivation method...sequencing-based lineage tracing Recently, gene expression in all cells of the developing zebrafish embryo..., relying on established reagents such as CRISPR/Cas9, Tol2, and the high-quality sequenced zebrafish ..., and their resistance (so far) to facile CRISPR/Cas9-mediated insertion of foreign DNA. Despite these...cells from 694 embryos over multiple stages and reconstructed the patterns of gene activity and cell divisions...