...near-matches to reduce risk of cleavage away from target sites (off-target effects). Ensure that the guide sequences...primers upstream and downstream of the sgRNA cleavage sites (“deletion band”; Figures 1 - 2 ). In the absence...sorted cells individually into 96-well round-bottom plates using sorter or by using limiting dilution at 30...conditions for each primer pair designed based on testing the bulk sorted cells. Run samples on 2% agarose... per well for a final volume of 150 μl. This facilitates screening by allowing a multichannel pipette ...100 μl each into two separate 96-well flat-bottom plates. Keep one plate to allow for clones to grow and... into DH5α E. coli cells and plate onto LB agar plates with the relevant antibiotic. Select multiple colonies...

...including: how closely homologous the off-target sites are compared to the on-target site, the specific...immediately adjacent to the nearly-homologous target sites. The mere presence of additional PAM sequences should...selection tool that should be helpful in minimizing or testing off-target DSB. Is it possible to target a single...summarize HR template vector design guidelines? A few notes below are considerations for designing HR donor....with PX330 plasmid, clone in your target guides, test run them and then select the best guide to co-transfect...site in the donor. For example, choosing target sites that span the knock-in gene. For making mutations...As a successful insertion will destroy the BbsI sites, a double digest should discriminate between positive...

...partial homology to additional sites throughout the genome. These sites are called off-targets and should...efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Scientific...Once possible PAM sequences and putative target sites have been identified, it is time to choose which... gRNA target sequence does NOT match additional sites within the genome. In a perfect world, your gRNA...examined during gRNA design. In general, off-target sites are not cleaved as efficiently when mismatches occur...

...function of zebrafish astrocytes. Knock-in tagging - Michel Bagnat lab. Targeting cassettes encoding fluorescent...Zebrafish Society (IZFS) - An organization that promotes and advocates for zebrafish research in the international...Morsch Lab. A vertebrate model to study ALS-linked aggregates in real time. Live imaging of astroctyes - Kelly...Resources Zebrafish Genome Project - Access the latest reference sequence assembly for the zebrafish genome...

...Institute Identifying Altered Epigenetic States and Drivers in Intestinal Carcinoid and Pancreatic Neuroendocrine... Cancer Institute Finding the Causes of Small Intestinal Neuroendocrine Tumors Ramesh Shivdasani Dana-...Dana-Farber Cancer Institute Epigenetic Regulators of Intestinal Endocrine Cells and Carcinoid Tumors Qiao Zhou...Zhou Harvard University Epigenetic Regulators of Intestinal Endocrine Cells and Carcinoid Tumors Bradley ...

...simultaneously with dual labeling when used with two substrates Can be used in live or fixed cells, and for internal...protein tags based on the acyl carrier protein. Substrates are derivatives of Coenzyme A (CoA). These tags... Please see the note below for information on substrates and synthases needed for use of these tags. Plasmids...product portfolio, including the CoA-derivative substrates and the ACP and SFP Synthases. Users may still... their own fluorescent or non-fluorescent CoA substrates by purchasing coenzyme A disodium salt from a...

...pyogenes GFP Vakoc U6>sgRNA(F+E) 59986 Other/Ciona intestinalis BsaI none S. pyogenes Christiaen p(U6a-BsaI-...vectors containing multiple guide RNA expression cassettes and a Cas9 nuclease/nickase expression cassette...the expression of multipe gRNAs. Csy4 cleavable cassettes; requires expression of Csy4 (eg Plasmid 53369...gRNA module vectors, multiple gRNA expression cassettes can be assembled using Golden Gate cloning or ...polycistronic glycine tRNA-gRNA genes (PTGs) and then PTG cassettes are assembled into a Cas9 expressing vector. Yang...to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR...sgRNAs Zebrafish Plasmids for assembly of gRNA cassettes using Golden Gate ligation into Tol2-based pGGDestTol2LC...

...nuclease operates as a dimer, cutting in the spacer region between two distinct ZF target sites. (B) A ...species almost as easily as correcting a typo in the latest draft of your dissertation. Well, that will probably...corresponding zinc fingers, potential targetable sites in your average genome are every 500 bp. TAL effectors... a T), but they still have potential targetable sites approximately every 35bp. Researchers are still ...

...sequences and gRNA target sites. The first deposited PITCh plasmids were tested by fusing EGFP-2A-PuroR ...plasmids in this CRISPR-Cas tagging system were tested by tagging transcription factors with FLAG in human...plasmids in the following articles provide PCR templates for amplification of the tag (eg GFP, Flag, YFP...elegans . This system uses a 10-day protocol, generates “clean” homozygous mutants with no co-integrated...

...Cell type-biased: Astrocytes - AAV-PHP.AX with GRE or transgenic animal Oligodendrocytes - AAV-PHP.eB with...digital PCR, in vitro and (when possible) in vivo testing, and full sequencing of the final viral vector ...LY6A-nonpermissive mouse strains. **AAV1-X1 has not been tested in rats. Abbreviations: AAV, adeno-associated virus...regulatory element; NHP, non-human primate. Image courtesy of the Gradinaru Lab and the Caltech CLOVER Center...

...bacteria, yeast, Drosophila, zebrafish, C. elegans, parasites, and Xenopus. Addgene CRISPR Resources CRISPR ...genome engineering. CRISPR Topic Page : Want the latest news on CRISPR? Experts cover CRISPR topics on ...promoting DSB formation. Delete NEW Cascade-Cas3 generates large genomic deletions. Insert NEW CRISPR transposases... Activate dCas9 fused to an activator peptide activates gene expression. Interfere dCas9 alone, or fused...

... TALE transcriptional activator target sites and then generates individual user-friendly graphical roadmaps... ( Sander et al., Nat Biotechnol. 2011 ) Rats ( Tesson et al., Nat Biotechnol. 2011 ) Human somatic cells...monomeric TALE transcriptional activator target sites and to simplify the construction of TALEN expression...

...inducible - VIP vasoactive intestinal peptide MGC13587, PHM27 VIPR1 vasoactive intestinal peptide receptor 1 ...immune response is triggered within the first few minutes of exposure to a pathogen and is the dominant defense...Chemokines recruit innate and adaptive immune cells to sites of inflammation throughout the body. Cytokines Cytokines...its cognate antigen along with co-stimulation activates the T cell, whereas T cell tolerance or anergy... NK cells, macrophages, dendritic cells, and granulocytes. TLR The toll like receptors (TLR) play a key...Chemokines recruit innate and adaptive immune cells to sites of inflammation throughout the body. Symbol Name...chemokine (C-C motif) ligand 5 D17S136E, MGC17164, RANTES, SCYA5, SISd, TCP228 CCL7 chemokine (C-C motif)...

...Quality Control Each lot of antibody undergoes testing, either in-house or through outside labs, to confirm...storing at -20 °C. Each antibody catalog item is tested for shipping stability by incubating at 37 °C for..., freezing at -80 °C, thawing on ice, and then testing for loss of potency against a non-treated control...recommended assays. If an antibody has passed stability testing, it may ship at room temperature. However, Addgene...

... easy assembly of one or more gRNA expression cassettes - Qi-Jun Chen. Cas9 and gRNA backbone for use...create plant expression vectors containing several cassettes and generate multi-construct transgenic plants...The ABRC collects, preserves, reproduces, and distributes diverse seed and other stocks of Arabidopsis ... (Link opens in a new window) - The initiative promotes interdisciplinary exchange, open technologies,...

...changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and ...hypermutation in a 100 bp window. This technique creates diverse populations of mutants for directed evolution...adenosine deaminase to catalytically dead Cas13b creates a programmable RNA editor that converts adenosine... transcription factor and other protein binding sites. ID Plasmid Gene/Insert Promoter Selectable Marker...

...The data is also used to determine recombination rates in FLEX/DIO/fDIO constructs. Endotoxin Endotoxin...sterility. Transducibility Some viral vectors are tested in vitro and in vivo for gene expression and/or...line was obtained from Takara, and is routinely tested for mycoplasma contamination using mycoplasma detection... 2 weeks post-thaw, cell culture supernatant is tested for mycoplasma contamination. To date, Addgene ...

...ID Plasmid Description PI Viral Preps Addgene distributes ready-to-use viral preps made from plasmids in...and targeted to the neuronal soma and proximal dendrites in a viral vector Hillel Adesnik 107708-AAV9 pAAV-hSynapsin...ChroME targeted to the neuronal soma and proximal dendrites and separated from nuclear mRuby3 by P2A in a ...and targeted to the neuronal soma and proximal dendrites under the control of internal ribosome entry sequence...recombination Ofer Yizhar Antibodies Addgene distributes ready-to-use recombinant antibodies made from...

...includes titering, purity assessment, sterility testing, and viral genome sequencing (VGS). In-stock AAV...Neuronal Tracing Rabies Virus-Based Serotype Serotype Testing Samples Small samples of control AAV vectors for...CAP-B10, CAP-B22 University of Florida Serotype Testing Panel for the Eye and Brain For enhanced transduction...

... All Antibodies All Antibodies Addgene distributes ready-to-use recombinant antibodies. These monoclonal...recommended applications based on our in-house testing and data provided by trusted outside labs – with... represent the official views of the National Institutes of Health. Need Some Help? Browse some of our...