... by the Rinehart lab: pCRT7 NT TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant...68292 C321.ΔA bacterial strain – Addgene #68306 pCRT7 NT TOPO E17TAG GFP plasmid - Addgene #68295 SupD plasmid...identifies a staggering ~250,000 phosphorylation events in the proteomes of higher mammals (2). How can... the importance of any of these phosphorylation events on the activity of a given protein? Classical methods...5). Optimization of this system defined three components required for translation (Figure 1): A tRNA ...the pSer-incorporated polypeptide. These three components were shown to effectively incorporate phosphoserine...inactive without requisite dual phosphorylation events on its activation loop, but can be made constitutively...

...efficiencies of multiplex editing at each site. A 75-nt human cysteine tRNA stood out from their initial ...array, DAP arrays use tRNA itself, as short as 75-nt, to both drive and process the tRNA-gRNA arrays, ...both efficiency and multiplexity, with these constraints? The Gao lab first thought to enable MBE by ...the DAP array, the Gao Lab packaged MBE and MPE elements for deliveries via adeno-associated virus (AAV...

... unprocessed or processed gRNA array (22 nt spacer with 30 nt direct repeat) mediating potent knockdown...

...CRISPR/Cas system makes use of 3 components, a guide RNA (gRNA) of about 125 nt that specifies the target, ...the mouse genome is made up of 3 x 109 nucleotides (nt), and encodes 23,000 or so genes. It would be great...the gRNA will seek out its target among the 3 X 109 nt of genetic content in the mouse genome and the Cas9...gRNA, Cas9, and donor oligonucleotide or plasmid components are brought together and microinjected into either...eggs. Or, to avoid handling embryos ex vivo, the components can be electroporated into the oviduct of pregnant...CRISPR is so efficient that you can inject the reagents directly into fertilized mouse eggs, circumventing...friends and colleagues while working with these reagents. Other than that, be nice to your microinjectionist...

... kb ~3.9kb crRNA/gRNA length gRNA: ~100 nt crNA: ~42 nt Requires tracrRNA? Yes No dsDNA cleavage...Cas9 and Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 Endonuclease...AAV transduction.  In most of Zetsche et al’s experiments, a single vector co-expressing Cpf1 and a crRNA...culture of mouse neurons and in vivo. For these experiments, cells were infected with a 1:1 ratio of two ...editing with CRISPR-Cpf1 is one of the latest developments in CRISPR technologies.  It’s a simple and effective...thoughts about Cpf1 multiplexing, leave them in the comments below.        References 1. Zetsche et al. "Multiplex...

...)  Editing window ~2 nt  85175  pBK-EE-BE3  SpCas9 (NGG)  Editing window ~2 nt  85176  pBK-YE2-BE3...BE3  SpCas9 (NGG)  Editing window ~2 nt  85177  pBK-YEE-BE3  SpCas9 (NGG)  Lower on-target activity than... than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity...Campbell’s lab added two new mCherry variants to the repository. These variants can be used as tools to learn... Podcast Segment The impact of KLF4 N-terminal variants on iPSC generation Deriving iPSCs (induced pluripotent...transposons, the Woltjen Lab compared different variants of the polycistronic cassettes and discovered ... developed a new AAV approach using regulatory elements that restrict expression to this cell type. To...

... made up of microtubules, actin filaments, and intermediate filaments. These structures give the cell ...GFP-cortactin Actin Filaments Cortactin EGFP Anna Huttenlocher 32907 pEGFP-rNFM Intermediate Filaments (neuronal...Intermediate Filaments (neuronal) Nefm EGFP Anthony Brown 11908 pEGFP-N1 alpha-actinin 1 Actin Filaments alpha-actinin...Actin Filaments Cortactin mCherry Christien Merrifield 36201 pLifeAct-mTurquoise2 Actin Filaments LifeAct...pLifeAct_mScarlet_N1 Actin Filaments LifeAct mScarlet Dorus Gadella 85055 pLifeAct_mScarlet-H_N1 Actin Filaments LifeAct ... Actin Filaments ITPKA mTurquoise2 Dorus Gadella 58473 pEGFP-C1 F-tractin-EGFP Actin Filaments F-tractin...mCherry-Lifeact-7 Actin Filaments LifeAct mCherry* Michael Davidson 54610 mEGFP-Lifeact-7 Actin Filaments LifeAct EGFP...

...antibody fragments consisting of two scFvs bound to a CH3 domain. Fab fragments - Fab fragments are created... called the antigen binding fragments (Fab). The tips of the Fab fragments contain highly variable regions...Affinity Reagents There are several alternatives to antibodies, ranging from antibody fragments (shown ...Structure Isotypes Antigen Specificity Other Affinity Reagents Handling Visualization Signal Amplification Application...Multiplex Assays Selection Validation Affinity reagents are molecular tools used in the lab to specifically...foreign bodies, are the most commonly used affinity reagents. Antibodies are Y-shaped proteins, naturally produced...interest is introduced to the animal, which then mounts an immune response to it. Once the immune response...

...down which reagents are appropriate for a given experiment. Make sure to check whether reagents are available...you are ready to start navigating the different reagents that are available for your particular experiment...genetic manipulations require different CRISPR components. Selecting a specific genetic manipulation can...dCas9-KRAB) or dCas9 gRNA(s) targeting promoter elements of target gene dCas9-KRAB is more effective than...such as dCas9-VP64) gRNA(s) targeting promoter elements of target gene Many different activators exist...cells), transfection with standard transfection reagents may be sufficient to express the CRISPR machinery...(e.g. primary cells), viral delivery of CRISPR reagents may be more appropriate. In cases where off-target...

...in monocot and dicot plants - Yao-Guang Liu. Plasmids for prime editing in plants (including rice and ...Addgene's plant resources: Find plasmids for use in plants. Plasmid...tools that were specifically designed for use in plants. These resources include tools for research using...useful toolkits and plasmid collections for use in plants. The tables below highlight plasmids spanning a...selection of plasmids expressing different functional variants of Cas9 and empty gRNA backbones. A few examples...- Yinong Yang. The MoClo CRISPR/Cas Toolkit for Plants includes CRISPR/Cas nucleases, base editors, gRNA...assembly for fast generation of multiple Arabidopsis mutants - Niko Geldner. CRISPR-Cas9 vector with GRF4-GIF1...

... regulatory elements on gene expression. Empty backbones for inserting regulatory elements before or after...secreted and more stable, making time course experiments possible, but its short luminescence half-life...commonly used to investigate the effect of regulatory elements, such as promoters, enhancers and untranslated...Conformationally destabilized Firefly luciferase mutants : A set of three sensors that are increasingly ...for split Firefly Luciferase complementation in plants. Bioluminescence Resonance Energy Transfer (BRET...Creating ORF-Firefly luciferase gene fusions in plants. Renilla luciferase under the control of a CMV ...white screening can be used for screening of transformants in cloning process. Lynne Postovit 119760 pSGDlucV3.0...

...multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements, and other Cas...scale edits by integrating segments of DNA flanked by specific repeat elements into the genome. CRISPR-associated...mechanism Engineered CRISPR systems contain two components: a guide RNA ( gRNA or sgRNA ) and a CRISPR-associated...mechanisms of CRISPR and some of the ways the CRISPR components are leveraged to achieve specific goals, such...the Cas enzyme. Basic Mechanisms of CRISPR The requirements for a CRISPR knockout assay are simple. You’...Cut) Multiplex Genome Engineering Many CRISPR experiments involve editing more than one gene. In these ...RuvC and HNH, can be altered to reduce cleavage events to a single cut (nick) or no cuts. Cas9 nickase...

...antibody components into plasmids and expressing these in bacteria, mammalian cells, yeast, plants, or insect...size. These recombinant antibody fragments can be used in experiments such as immunoprecipitation and ...antibodies as Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. Functional diversification...microscopy. Two commonly used plasmid-based antibody fragments are scFvs and single-domain antibodies (also known..., scientists often use plasmids encoding scFv fragments that bind to the GCN4 peptide, from the popular...the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods. 2000 Aug ...dimerisation and detection of recombinant antibody fragments in the form of scFvs A simple vector system to...

...Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales APW, Li Z, Peterson RT, Yeh J-...Addgene. Kleinstiver BP, Tsai SQ, Prew MS, Nguyen NT, Welch MM, Lopez JM, McCaw ZR, Aryee MJ, Joung JK...The number of off-target cleavage events for the VQR and VRER variants is similar to wild-type SpCas9, ...reported the development of near-PAMless Cas9 variants. These variants, named SpG and SpRY, can target NGN PAMs...the number of CRISPR reagents continues to grow, so too will the number of reagents available through Addgene...limitation: 1) the use of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9...positive selection screens in bacteria to identify mutants of S. pyogenes Cas9 that were able to cleave target...

...prepared in large amounts and frozen for future use or directly ligated with the annealed 24 nt oligos to generate...the spacer(20nt) on pEcgRNA plasmid as only two 24 nt oligos are required and the BsaI linearized pEcgRNA...not function properly in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF... This would result in the lack of recovered transformants. Our speculation motivated us to update the ...system While the above systems are great for experiments that require only single chromosomal integration...

... Guide for more information about: Lentiviral components 2nd versus 3rd generation lentiviral systems ...feature of lentivirus used in research is that the components necessary to produce an infectious viral particle...called packaging and envelope plasmids) encode components of the viral capsid and envelope and are used...transfer plasmids along with additional packaging components Trono 63586 psPAX2-D64V 2nd Packaging For making...that encodes genetic material that the researcher wants delivered to specific target cells. This genome ... See article for more cre-lox shRNA expression variants. Jacks 19319 pLJM1-EGFP 3rd CMV-driven EGFP fusion... 11619 pLB 3rd Modification of pLL3.7; Genetic elements known to prevent epigenetic silencing were added...

...point in bacterial growth? Can I produce large amounts of this protein and study it in vitro ? These, ...resources required for the production of large amounts of protein or high copy number plasmids. Whether...fluorescent protein variant (CyPet) optimized for FRET measurements with a yellow fluorescent protein variant (YPet...fluorescent protein variant (YPet) optimized for FRET measurements with a cyan fluorescent protein variant (CyPet... pCri System Various Plasmids T7/lacO and lacO variants that can be expressed in B. subtilis Various combinations...constituitive promoters and translation initiation elements that can be used to drive gene expression at a...Plasmids Reporter plasmids can be used to detect events and molecules in and around a bacterium. Plasmids...

...for many years and lead to a major burden on the patients, their families and caretakers, and the health...general. The required care for these millions of patients results in billions of dollars spent each year...changes in behavior and emotion, uncontrolled movements, memory deficits, as well as difficulty speaking...after a person's mid-sixties. The early-onset form accounts for less than 10 percent of all cases of Alzheimer's...plasmids expressing wild type or mutant ORFs, gene fragments, and gRNAs for genes linked to neurodegenerative...discovery and advance development of diagnostics and treatments for Alzheimer’s disease and related disorders...foundation offers curated information on tools and reagents (Link opens in a new window) such as huntingtin...

...The RESOLUTE mission is to develop data and reagents to boost the research focused on Solute carrier ...Union. The RESOLUTE mission is to develop data and reagents to boost the research focused on SLCs. Through...open-access ethos’ to the results, techniques and reagents with the highest-possible quality of research ...knowledgebase that features, among others, genetic variants and disease associations. Plasmids This plasmid...libraries are useful tools for genetic screening experiments. These libraries target the solute carrier family...feedback form . If you have further questions or comments you can also contact them at [email protected]...

...payment, and billing. Industry Material Transfer Agreements Most Addgene orders must be accompanied by a ...protection for material providers. Addgene’s deposit agreements allow Addgene to distribute plasmids on behalf...behalf of the depositing institutions. These agreements indicate the language which we are allowed to use...use for distribution. Please see the Sample Agreements on our Technology Transfer Information page for ...authorized signatory is someone who can execute legal documents on behalf of the recipient institution. Once the...addgene.org . After we review and approve the MTA documents, your order will be processed and shipped. Industry...