...they need to use them in their own work, it also increases the visibility of both you and the original depositor...work later. We also hope that this feature will decrease redundancy. If you modify a plasmid from Addgene...out there Beyond these 3 big pushes, we try to increase your work’s visibility in a few other ways. Every...

... this technology.” GeCKO library for genomic screens Using lentivirus as the delivery vehicle, Neville...to enable both negative and positive selection screening. They and their colleagues including Feng Zhang...at a time,” Wang said. “It was very difficult to screen in a pooled fashion or to go after thousands of...expression. The Yusa lab has used their library to screen for genes involved in bacterial toxin resistance... is now possible to conduct systematic genetic screens in mammalian cells,” David Sabatini, a senior author...Library Reference page.    References: Genetic Screens in Human Cells Using the CRISPR/Cas9 System. Wang... (PubMed). Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem O, Sanjana NE, Hartenian...

...drive expression of Cre-mCherry and a second promoter to drive expression of a Cre-dependent fluorescent...will only result in cells where Cre-mCherry is also expressed. If Cre-mCherry expresses in photoreceptors...photoreceptors and horizontal cells, and Cre-dependent-GFP expresses in horizontal cells and ganglion cells, only...horizontal cells transduced by both AAVs would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters...different design strategies: Strategy #1 (ProA): Create promoters with sequences upstream of the start ...cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved sequences...high cell-type specificity.  Strategy #3 (ProC): Create promoters by repeating mouse cell type specific...

...AAV-GfaABC1D-Cre-4x6T AAV5 Recombinases Stanley Thomas Carmichael New viral prep pAAV-EF1a-fDIO-Cre AAV5 ...Collection. These preps drive expression of either Cre recombinase or a fluorescent marker in the various...Recombinases Esteban Engel New serotype pAAV-hSyn-Cre-P2A-dTomato AAV PHP.eB Recombinases Rylan Larsen ...Larsen New serotype AiP13778 - pAAV-AiE0450h-minBG-iCre(R297T)-BGHpA (Alias: CN3778) AAV PHP.eB Recombinases...viral prep AiP13779 - pAAV-AiE0779m_3xC2-minBG-iCre(R297T)-BGHpA (Alias: CN3779) AAV PHP.eB Recombinases... New viral prep AiP13781 - pAAV-AiE0452h-minBG-iCre(R297T)-BGHpA (Alias: CN3781) AAV PHP.eB Recombinases...

...biological process. These screens provide an alternative to genome-wide RNAi screens, which although highly...similar pitfalls exist for CRISPR screens and how best to design these screens to controls for flaws. Recently...CRISPR knockout screen to analyze the following unanswered questions about CRISPR screen design. Unanswered...CRISPR/Cas9 screen design Are non-targeting guides an appropriate control? Many CRISPR screens use non-targeting...optimal conditions for CRISPR knockout screens, the Bassik lab created a human knockout library that has 10...library was validated via a growth screen and a ricin toxicity screen since essential and non-essential...The library performed well in both screens, with the growth screen having a 1% false discovery rate and...

...gain-of-function screening when combined with a compact pooled sgRNA library. Our SAM-mediated screens exhibited...gene activation in mind. “We had not managed to create a system to allow us to reliably activate essentially...the next step in high-throughput, genome-wide screening,” Konermann said. “If you want to activate all...and make sure it’s working. You can only do that screen with a system you are really confident about working...her colleagues demonstrated the new system by screening for genes that, when turned on, make melanoma ...CRISPR library enabling genome-scale, knockout screening). As the Zhang lab's Nature paper reports, “Here...validation of the top 10 hits.” Two-way CRISPR screening In October, Jonathan Weissman’s lab introduced...

...DreO-bGHpA 121675  AAV9  pAAV-EF1a-fDIO-Cre (FLP-dependent CRE) 55634  AAV1  pAAV-EF1a-mCherry-IRES-... constitutive (pAAV-Syn-Archon1-KGC-GFP-ER2) and cre-dependent version (pAAV-Syn-FLEX-rc [Archon1-KGC-...membrane-embedding voltage detector. Find Archon1 and its cre-dependent version at Addgene! Fluorescent protein...

...these issues by creating immortal antibody-producing cell lines (hybridomas) that secrete antibodies in ...Neurotechnologies® (BRAIN) Initiative, will allow Addgene to create a much needed open-access recombinant antibody/...School of Medicine on this grant. Dr. Trimmer has created thousands of high-quality antibodies/nanobodies...reagent batch. To supplement this, Addgene will be creating a crowdsourced Data Hub (built off of the concept...resource to life,” says Dr. Trimmer. But in order to create such a resource, we need your help. You can participate... Director. “NABOR provides a new model for the creation and acquisition of affinity reagents and will ...funding laboratories, such as Dr. Trimmer’s lab, to create recombinant antibodies for use in neuroscience ...

...tested iTOP in a series of primary cell types using Cre and various loxP reporters. In both mouse and human...potential application of iTOP Having tested iTOP using Cre-lox recombination, D’Astolfo et al. were eager to...protein expression has a long-lasting effect, such as Cre- or Cas9-mediated recombination. Both primary hESCs... cre-lox...

...researchers injected Cre-expressing AAV-5 into the DRN of a transgenic mouse harbouring a Cre-dependent IL-1...injected AAV2 particles into a Drd1-Cre transgenic mouse to deliver Cre-dependent expression of  Channelrhodopsin...-1 beta overexpression cassette. Expression of Cre in these neurons turns on overexpression of IL-1 beta...2014). Scientists from the University of Geneva (Creed et al., 2015) used optogenetics to support these...transmission from the mPFC projections to the NAc and decreased the locomotion of the animal. This confirms the...behavior in rats in the forced swim test as seen by increasing the ratio of time spent actively swimming versus...described here is only a small selection of the increasing number of studies using viral vectors in behavioral...

...Expression of hM4D(Gi)-mCherry from this virus is cre-dependent, meaning that once injected, the DREADD...DREADD will only be expressed in cells that express cre recombinase. Typically, this feature is used to attain...cell-specific expression in transgenic animals, where cre expression is restricted to a particular cell type...the first time, the Roth Lab recommends verifying cre expression prior to use. The lab additionally recommends...expressing cell lines, which can then be used both for screens (using pooled CRISPR knockout libraries) and individual...Secondly, you can use a LentiCas9-Blast viral prep to create a single stable Cas9-expressing cell line and then...monoclonal Cas9-expression line prior to performing a screen or even a single gene knockout. Top AAV: pAAV-hSyn-DIO-hM4D...

... For example, a researcher can deliver Cre-dependent TVA to Cre-expressing neurons in a transgenic mouse...Addgene Blog AAVs CREATed for Gene Delivery to the CNS and PNS Advanced Uses of Cre-lox and Flp-FRT - ...viral protein EnvA on its surface will only infect Cre-positive, TVA-expressing cells (Osakada et al., 2013...

... the absence of Cre. mCherry expression alone was detected. pAAV-Ef1a-mCherry-IRES-Cre was a gift from...were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634-AAVrg) alone at 1.7E6 viral genomes (vg)/cell...) alone at 1.1E6 vg/mL, or both. Two days later, Cre-dependent GFP expression was detected with direct...

...Addgene depositors have developed heat shock-inducible Cre and Cas9 for easy genome engineering in species like... Plasmids 101: The Promoter Region Plasmids 101: Cre-lox Light-Switchable Two-Component Systems for E....Tetracycline (Tet) On Expression Systems at Addgene Cre-lox Guide and Plasmids     ...cell growth media with glucose decreases cAMP and represses pBad, decreasing promoter leakiness.   Other ...

...interneurons can be labelled with an AAV1 Cre virus combined with a Cre-dependent AAV9 tdTomato virus in the...learn more about the AAV Data Hub, and hear the incredible AAV talks given by Connie Cepko and Ian Wickersham...

...control when you turn off expression for good, the Cre-Lox system offers inducible yet permanent gene excision...can be excised by intentional introduction of the Cre recombinase. This is a time-tested system but does...some permanent knock out plasmids? Check out our Cre vector and Murine LoxP vector.   Turn it off for ...!  Turn it on Turn it on forever Permanently increasing or turning on expression of a new protein is ... gRNA, to your gene of interest to temporarily increase expression. There are several flavors of activators...

...labeling Increased neuronal expression using Cre-mediated AAV Targeted Evolution method (M-CREATE) dCas9...-4.   Increased neuronal expression using Cre-mediated AAV Targeted Evolution method (M-CREATE) by: Brian...multiplexed version of the Cre-mediated AAV Targeted Evolution method (M-CREATE), in which one applies both...variants that display increased neuronal expression in the brain while having decreased expression in the ...zebrafish as a model organism, the Parton Lab recently created a flexible system that makes use of pre-existing...Image from Goertsen D, et al., 2022.  Find M-CREATE plasmids here!  Goertsen D, et al. Nature Neuroscience...

...doxycycline, but it can also be regulated spatially with Cre-recombination. Read the paper in Frontiers in Molecular...Averof lab. It’s an alternative to the conventional Cre or Flp recombinases used to excise markers. It has...Cas12a inhibitors Using a bioinformatics and a screening approach, the Jennifer Doudna lab identified three...

...open-reading frame (DIO). Upon coexpression with cre recombinase (either from a mouse line or viral co-injection...type specificity dependent on the promoter of the cre recombinase while still taking advantage of the robust...an appropriate mouse line expressing a particular cre, viral expression of your opsin is no longer required...tracing with fluorescent proteins in nontransgenic and cre driver mice. Current protocols in neuroscience / ...: PMC4242517.  7. Madisen L, et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced... us to show causality in neural circuits with incredible temporal and spatial precision (I’m not going...optogenetics has already been proven. Therefore, a concrete, well defined, and robust behavioral output is...

...often made Cre-dependent. If Cre-dependent vectors are used in transgenic animals that express Cre in only...labelling the subset of infected cells that also express Cre. Another advantage of AAV vectors is that there are... traced neurons with an AAV9 vector expressing a Cre-dependent fluorescent marker. Injection into a conditional...conditional mutant mouse brain that expressed Cre recombinase only in pyramidal cells of the hippocampal...PMID: 4107258. Lo, L., and Anderson, D.J. (2011). A Cre-dependent, anterograde transsynaptic viral tracer...

... short hairpin) and CRUSH (Conditional RUSH) use Cre-mediated recombination to turn on or off the expression...a dual-color RNAi “sensor” mouse strain in which Cre expression causes the induction of both DsRed fluorescence...Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown in eyes and... recombination events at the genetic level. Upon Cre/loxP recombination, each transgene expresses one ...Flp recombinase/FRT system in place of Brainbow’s Cre/loxP, allowing for simultaneous use of the two systems...SUMO tags in the FP sequence for separation from Cre-based Brainbow-expressing cells. Autobow plasmids... all-in-one versions which express self-excising Cre recombinase from the same transgene as the Brainbow...

...sensors include syn-jYCaMP1s (slow variant), the Cre-dependent syn-FLEX-jYCaMP1s, and the axon-targeted...syn-FLEX-axon-jYCaMP1s. Find these AAVs at Addgene Cre-dependent EYFP AAV in the new serotype PHP.V1 that...Find this AAV at Addgene New serotypes for the Cre-dependent Flpo recombinase pEF1a-DIO-FLPo-WPRE-hGHpA...biotechnology tool to optimize another. The authors have created a yeast-based directed evolution approach with ...your studies.  Find the plasmids to set up your screening systems at Addgene! Schematic of the evolution...following a DNA miniprep protocol!  First, the authors created the pMAK backbone by modifying pMAL-T-Avi-His/BirA...exported to the periplasm of bacteria where it can be secreted into the supernatant. Four plasmids containing...

...in CRISPR genome editing.   2. Plasmids 101: Cre-lox Cre recombinase can be used to generate inversions...community members. In addition we’ve experienced incredible growth with our readership expanding from ~10,000...next revolution in biological research. We’re incredibly happy to see that one of our career posts has...protein of interest. 4. Plasmids 101: Blue-White Screening Getting a lot of empty vector contaminants in ...Addgenie Jessica Welch explains how Blue-White screening could be a potential solution. 5. Cpf1: A New ...

... the expression of a Cre recombinase contained within the knock-in. Including Cre-mediated marker excision...kilobases of additional sequence being introduced. Cre-mediated excision of the selective cassette minimizes...experimental manipulation. In some cases, injection of Cre is required, however the development of the self-...variable amounts of experimental manipulation and screening.  A co-selection strategy for CRISPR editing in... with inefficient sgRNAs in my initial direct screening efforts, which were similar to the approach detailed...low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering rare editing events...conditional-lethal mutation to identify edits with minimal screening effort. Selecting for repair of a lethal mutation...

...recombinase that can be used in combination with Cre for intersectional control of specific neuronal populations... We now offer a Flp-expressing AAV, as well as a Cre-dependent Flp-expressing AAV. See our recombinase...

...CAG-FLEX: CAG promoter, FLEXed for expression in Cre-expressing cells hSynap: Human Synapsin-1 promoter...hSynap-FLEX: Synapsin-1 promoter, FLEXed for expression in Cre-expressing cells. hSynap-FLP: Synapsin-1 promoter...they created three affinity variants – A184S (increased affinity for glutamate), S72A (decreased affinity...as: iGluSnFR3 v82: increased affinity and slower kinetics iGluSnFR3 v857: decreased affinity and faster... forms of neuronal signaling and regulation. To create iGluSnFR, the Looger lab wedged GFP between two...to glutamate, conformational changes lead to an increase in fluorescence intensity from the inserted GFP...iGluSnFR, the Looger lab swapped eGFP for sfGFP to create SF-iGluSnFR. This improved variant has higher expression...

...piggyBac™, you could use recombinase technology (Cre/loxP) to remove the donor cassette. While this method...mutants [7], it is generally not as efficient as Cre recombinase. However, the use of negative selection...likely due to DNA toxicity. More importantly, unlike Cre recombinase, the PBx is highly specific, and has ...applications, including transgenesis, gene-trap screens, and gene editing. How does the piggyBac™ system...of the major uses of the piggyBac™ system is to create stable transgenic cell lines. The traditional method...site-specific nuclease (such as CRISPR/Cas9) to create a double-stranded DNA break. After DNA cleavage... editing? There are primarily two other ways to create specific point mutations by gene editing. First...

... be achieved using specialized promoters, Flp or Cre dependent gene switches, and serotype selection. ...cell-specific gene expression and viral infection. Cre or Flp dependent switches control gene expression...

...targeting inhibitory or excitatory synapses or that are Cre-inducible, researchers can explore synaptic integration...preps at Addgene hSyn1-SIO-stGtACR1-FusionRed is a Cre-dependent, soma-targeting inhibitory channelrhodopsin...brighter in worms than split-sfCherry3. It was created by Maria Ingaramo, Jerome Goudeau, Cynthia Kenyon...Addgene The Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA) Knockout Libraries express...

... floxed dCas9 so that it may later be excised by Cre recombinase. Addgene has several great options: pLV...T2A-GFP for removable dCas9 expression and pLM-CMV-R-Cre. Selection for cells stably and highly expressing...are dioxygenases that oxidize 5-methylcytosine to create rare DNA bases that are recognized by distinct ...

...useful tool for marking and tracking cells is the the Cre/lox recombination system. A researcher can flank ...expression is turned on or off in cells expressing Cre. The result of a system like this is a mosaic of ...respectively. The permanent labelling of cells using cre-lox is advantageous when compared to photoactivable... In the past 15 years efforts have been made to create transgenic mice expressing fluorescent protein ...wavelength as it has been shown that even a small incremental change of the excitation wavelength can significantly...called Brainbow, the Ubow mouse strain has been created to fate map Langerhans cells and follicular dendritic...microscopy and new constructs have facilitated the creation of mouse lines that can be used in fate mapping...

...bacteriophage integrase phiC31. The popular UAS/Gal4 and Cre/Lox gene regulatory systems have also been successfully...zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments." Methods in cell biology...two revolutionary large-scale random mutagenesis screens produced ~1,500 zebrafish mutants that are still...deactivation tools for zebrafish Random mutagenesis screens provided a major toehold into understanding how...et al., 2019). Generating zebrafish transgenics Creating transgenic organisms by introducing foreign DNA...one-eyed pinhead mutant from the random mutagenesis screens. Microscopy-based lineage tracing can help illuminate...

...More About Designing gRNAs Against Your Favorite CREs Here Additional Resources on Addgene.org Find ... cre... to make out the details. To increase the resolution, the Xu lab created a dCas9-based CAPTURE (CRISPR... smaller pieces. Sticky ends created are then ligated together to create loops of DNA. This is key to ...that, when bound by transcription factors, can increase a gene’s transcription. Sometimes enhancers are...method The original CAPTURE method requires the creation of a stable cell line that co-expresses three ...DNA into smaller pieces, and proximity ligation creates chimeric circles of DNA that are distant linearly...

...Cell type-specific manipulation with GFP-dependent Cre recombinase.” Nat. Neurosci. 18(9) (2015): 1334-1341...

... Ace2N-mNeon-A122D and Voltron2 are available as Cre-dependent AAV1 vectors and are suitable for extended...found that administering these AAVs with a dilute Cre-expressing vector resulted in a sparsely-labeled ...CRISPR screens By Emily Bentley Building on their CROPseq method for optical pooled CRISPR screening, Paul...Multiplexed perturbation and decoding in pooled CRISPR screens Improved prime editor PE7 Antibodies to facilitate...multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi (Walton et al. 2024). This...enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors for CROPseq-multi...multiplexed perturbation and decoding in pooled CRISPR screens. bioRxiv 2024.03.17.585235; doi: https://doi.org...

...(Guy et al., 2007). They accomplished this using Cre-Lox mouse models in which the endogenous MECP2 gene...excision of the Stop cassette using tamoxifen-inducible Cre not only halted the neurological damage but completely...two MECP2 labs (Gail Mandel and Stuart Cobb) to create the Gene Therapy Consortium. Leveraging their combined...

...formation in iPSC-derived and iPSC-transplanted mice. Cre-deletable or Tet-inducible lentiviruses address some...considered safe for clinical applications. Excisable Cre-lox lentiviral vectors: This approach is the same...standard lentiviral vector, but transient expression of Cre recombinase allows for deletion of the inserted transgenes...insertional mutagenesis. Random integration also creates heterogeneous iPSC cell lines, which can complicate...insertional mutagenesis and random integration creates variation between iPSC clones. Retroviruses are...which is removed by culturing cells at 38℃, but screening cells for the presence of transgenes is still ...

...Systemic capsids packaging Cre-dependent transgenes can also be paired with Cre-expressing transgenic mouse...with appropriate cell type-specific promoters or Cre lines. Spatial transcriptomics suggests that AAV-...Huber, N., Pasca, S. P., & Gradinaru, V. (2016). Cre-dependent selection yields AAV variants for widespread...Deverman, B. E., & Gradinaru, V. (2020). Multiplexed Cre-dependent selection yields systemic AAVs for targeting...have developed techniques like CREATE (Deverman et al., 2016), M-CREATE (Ravindra Kumar et al., 2020),...primates (NHPs). These engineered capsids have increased transduction efficiency in the CNS, with greater...

...But CRISPRing typical mouse models, including creating Cre-dependent conditional alleles, has remained ...fluorescent proteins (EGFP, mCherry etc.), recombinases (Cre, Flp etc.), and tetracycline drug induction systems...plasmid pmScarlet_C1! After creating the T7-DNA template, you'd use ivTRT to create the ssDNA construct as ...CRISPR genome editing has made it easier to create knockout alleles in a variety of species, including...repair template is used. The original design for creating a conditional allele, or floxed mouse, from Yang... and inject the ES cells into a mouse embryo to create chimeric mice. This laborious process takes ~8-...product as a template for reverse transcription to create your ssDNA. Note that the T7 promoter will not ...

...Combinations of classical genetic tools, for example Cre/loxP or Flp/FRT, with AAV vectors are common approaches... strategies Combining different approaches can increase the specificity of cell targeting even further...delivery of AAV at the injection site results in increased specificity of the targeted cell population.  ...are also developing ways to improve delivery by increasing transgene cargo capacity, enhancing anterograde...