...CRISPR/Cas9 and AAV Viral Vector Elements Additional Resources on Addgene.org Find AAV Vectors for Your Research...capacity, AAV vectors were suitable for our needs for the following reasons: Safety: Since AAVs are nonpathogenic... cells an AAV can infect) can influence an AAV’s value greatly. Applying this mantra to AAV selection,...viral-vectors/aav/aav-guide. Where in the tissue/what cell type? In the CNS, for instance, there are marked... I knew little about adeno-associated Viral Vectors (AAVs) before starting my graduate program at the ...types in locomotion. We have relied heavily upon AAV vectors to deliver chemogenetic/optogenetic constructs...nonpathogenic, we are able to use AAV vectors without specific facilities (Biosafety Level-1). Other possible...

...single gene knockout. Top AAV: pAAV-hSyn-DIO-hM4D(Gi)-mCherry Our current AAV inventory consists almost... is a dual-vector system, meaning it is typically used in conjunction with a second vector that delivers...alternative single-vector systems typically express Cas9 and a gRNA from the same plasmid. LentiCas9-blast...constructs (i.e., using a dual-vector system as opposed to a single-vector system) has several benefits.... transductions with a single-vector system). Tips for using this plasmid Cas9 expression is typically ...of the human synapsin promoter. Purpose of this AAV pAAV-hSyn-DIO-hM4D(Gi)-mCherry can be used to induce...ready-to-use lentivirus and adeno-associated virus (AAV) to scientists around the world. We began with only...

...together (~4.2 kb) into an AAV vector is challenging due to its packaging capacity of AAV (~4.7 kb). While this...problems with Cas9 delivery. Adeno-associated viral vectors (AAV) are commonly used for in vivo gene delivery...packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity but necessitating...co-infection of two AAVs. Cas9 orthologs: shorter, but just as potent and specific? The two AAV strategies described...for in vivo studies. Testing AAV-SaCas9 in vivo To test the efficiency of AAV-SaCas9 in vivo, the Zhang ...not increased compared to a control AAV-GFP. Not only did the AAV-SaCas9-sgRNA constructs mediate genome...proteins with AAV, at just 1,108 and 986 amino acids, respectively, the size of both is within AAV’s packaging...

...also called AAV2-retro) Looking for more viral vectors? Browse our in-stock viral vectors or consider ...purification, and titering. Our newest viral vector service, AAV Packaged on Request  has seen a fantastic...consider our AAV Packaged on Request service, which offers over 3,000 eligible transfer plasmids and five different...Addgene's Data Hub What is a Virus (and a Viral Vector)?  AAV Variables that Matter...serotype pENN.AAV.CMVs.Pl.Cre.rBG AAV5 Recombinase James M. Wilson New serotype AAVrg = AAV retrograde...ready-to-use viral vectors in the Addgene repository  Name Serotype Category Depositor  Notes pAAV-CAG-emiRFP670...Brain Science New viral tool pGP-AAV-CAG-FLEX-jGCaMP8m-WPRE AAV5 Biosensor GENIE Project New serotype...

...or on our hot plasmids webpage. Listen to the entire Hot Plasmids podcast! Hot plasmids covered in this...the Tol 2 Gateway-Compatible Toolbox  Hot Plasmid 2: AAV Vectors for tageting GABA-ergic interneurons. Listen... Listen to Our Segment on these new AAV Vectors Hot Plasmid 3: mCyRFP1 - a new, monomeric, and cyan-excitable... of our Hot Plasmids podcast series, you'll learn about new red fluorescent proteins, AAV tools for targeting...nervous system, and vectors for zebrafish engineering. You can find additional hot plasmids in our newsletter...this segment Hot Plasmid 1: The Tol2 Gateway-Compatible Toolbox for quickly generating zebrafish transgenic... protein. Listen to Our Segment on mCyRFP1 Hot Plasmid 4: mScarlet - a new, bright, and monmeric red fluorescent...

...guide to viral vectors Find all of our viral vector blog posts Download our viral vectors 101 eBook Additional...ways to improve viral vector production. Whether redesigning viral packaging plasmids, investigating different...membrane When packaging adeno-associated viral vectors (AAV) not all serotypes are equal; while some serotypes...improve their AAV yields. Given the wide range of downstream applications scientists rely on our AAV preparations...adapted by vector cores and laboratories alike as an easy and inexpensive method to improve AAV yields. ...pressure on the production of retroviral vectors: Enhancement in vector stability." Biotechnology and bioengineering...To our knowledge the effect of hyperosmolarity on AAV production yields had yet to be examined. Increased...

...lab are now available as ready-to-use AAV vectors in serotype AAVrg.   ...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...highlights from recent CRISPR plasmids. To find all of the CRISPR plasmids available from Addgene, head...and putative pre-synaptic terminals with one vector. AAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby from Liqun...cortical interneurons. We now provide ready-to-use AAV vectors expressing GCaMP6f with this promoter. Image ...dendritic neurites from axonal neurites with AAV vectors expressing cytoplasmic targeted tdTomato and ...

...questions!  AAV Prep and Technique Q1: Is there any way to deliver a long gene (>4.5 kB) by AAV? A: One potential...compared to just one vector.   Q2: Do you think it is possible to make an efficient AAV prep for a 4.2 kB...the CMV promoter using the AAV-BR1 capsid? A: It is our understanding that AAV-BR1 has the same genome size...served by a dual vector, split intein approach.   Q3: What’s the best method for titering AAV? Is there a ...samples within a project. Targeting Your AAV Q4: Are there any AAVs that have successful transduction in ...different AAV particles into the same cells in vivo? A: This will be highly dependent on vector dose. The...Tips for a first time AAV user Resources on Addgene.org Find Adenovirus Plasmids Adenovirus Guide Page...

...choosing AAV or lentiviral vectors in our previous blog posts.  Viral vector injection Viral vectors allow...expression vector and the vector particles are delivered into the brain region of interest. AAV or lentivirus...lentivirus?  Both AAV and lentiviral vectors are used for intracerebral injections. AAV particles are smaller...lentiviral vectors are superior packaging vectors for larger genes - they can hold up to 8 kb, while AAV can ...viral vectors blog posts Find tips for preparing AAVs View our Beginner's Guide to Viral Vectors Resources...possible outcomes. Typically, AAVs have to be minimum around 1012-1013 vg/ml (vector genomes per milliliter)...using viral vectors By modulating neuronal gene expression and activity, you can use viral vectors to study...

...transfection of single plasmids vs pooled plasmids; for multiple edits, single plasmids are generally more...expression vectors which are often more difficult to transfect or package into viral vectors. Cas9 multiplexing... from crRNA expression plasmids resulting in smaller, more streamlined vectors which are easier to use...transduction, and AAV transduction.  In most of Zetsche et al’s experiments, a single vector co-expressing...transfected on a single plasmid. This is versus 2.4% of cells transfected with a pool of plasmids that contained...contained one crRNA per plasmid plus a Cpf1 expression vector.  See the graph in figure 2 for a comparison of...array that can be processed by Cpf1. Lastly, an AAV vector was used to multiplex edit 3-4 genes in a primary...

... Biosensor AAV Viral Vectors 101 eBook Resources on the Addgene blog An Overview of AAV Encoded Calcium...engineered into viral vectors, so if you want to use them, you’ll need to use the vectors (or redesign the ... to achieve in whole organisms, but AAVs make it possible. In AAV systems, different serotypes can be ...gamma retrovirus, or AAV as your vehicle, we’ve got you covered with packaging plasmids! References and ...genetically encoded, and luckily, we have pre-designed plasmids for these systems! Ion sensors One common biosensor...mechanisms. For one, many viral platforms, such as AAV or retrovirus, can either permanently integrate and...

... If there’s a plasmid or AAV tool that you can’t find, you can always suggest a plasmid to us, request... virus (AAV) and lentivirus are now available from Addgene. While super useful, these vectors require ...Addgene's Viral Service Find Retrograde AAV Browse Optogenetics Plasmids ...want to get into the field. Find Optogenetics Plasmids for Use in Neuroscience Research Neuroscience ...to generate new knockout mice, using adenoviral vectors to deliver IGF receptors to the brain, and probing...likely someway you can apply it in neuroscience. AAV tools from Addgene are already having an impact on...400 who signed up for our newsletter and a free plasmid) were very excited about our new viral services...

...largely precludes delivery by adeno-associated vectors (AAV). This issue was recently circumvented by the... construct for generating the SPH mouse. (B) AAV plasmids used to express CRE under neuron-specific promoters...individual neuronal populations.  The Yang Lab’s AAV plasmids used to express Cre under neuron-specific promoters...comprehensive CRISPR guide Find CRISPR plasmids and resources Find CRISPRa plasmids ...including transgenic mice, neuron-optimized viral vectors, and high-throughput screening approaches. Here...genes can be relatively laborious because multiple plasmids/viruses are needed to deliver all the protein ... inducible Cre recombinases can be introduced by AAV or by mating with Cre-expressing mice. Figure...

...transfer plasmid), the envelope (Env) gene on a separate plasmid (envelope plasmid), and the polymerase ...different plasmids. For a retroviral system this looks like the genetic cargo in one plasmid (the transfer...Gag structural protein on yet a third plasmid (packaging plasmid). Viral production then proceeds by transfection...and adeno-associated virus (AAV). The least risky of these four viruses is AAV – it is not known to cause...Viral Plasmids Which Type of Virus Should I Use? Resources on the Addgene blog Viral Vectors 101: ...integrate at chromosome 19q13.4 qtr. (AAVS1)). For these reasons, AAV is usually classified as a BSL-1 safety... transfection of all three plasmids into a producer cell line, such as HEK-293T, production of viral particles...

...Search for AAV vectors to use with your research. Visit Our AAV Guide Page for info on AAV components,...transfer to the rat CNS: AAV PHP. EB vector dose–response and comparison to AAV PHP. B." Gene therapy 25.5...nervous system (PNS) targeting AAV  Get 1st time AAV user tips from an AAV rookie  Learn about some considerations...stops viral vectors, such as adeno-associated virus (AAV), from reaching their targets. The AAV9 serotype...candidate capsids.  Are you interested in using AAV-PHP.B vectors? See Supplementary Table 3 in Huang et al....considerations when using AAVs Learn more about using AAV as a tool for gene expression in mammals. Resources...components, common uses of AAV, and much more! Find ready-to-use AAV from Addgene or check out protocols for packaging...

...Production of both AAV and lentivirus requires three plasmids — a transfer plasmid containing your gene...transfer plasmid. The transfer plasmids have different packaging capacities depending on the virus. AAV has...baculovirus plasmid. Production of baculovirus requires only two plasmids — your transfer plasmid and the ...Viruses AAV and lentivirus Baculovirus; can produce AAV and lentivirus if needed # of plasmids Three ...resources on the Addgene blog Viral Vectors 101: Producing Your rAAV AAV Titers: Where Do They Come From ...gene of interest plus two packaging plasmids (three packaging plasmids for 3rd generation lentivirus production...production. AAV and lentiviruses can be produced in insect cells, but they require cloning specific AAV or lentiviral...

...our viral vector blog posts Resources at Addgene.org Find AAV plasmids Find ready-to-use AAV viral preps...fluorescent GFP marker (CatCH-GFP). The lab deposited AAV plasmids containing the active promoters at Addgene. ...Performance of synthetic promoter AAVs in mice All of these synthetic promoter AAVs were first injected into the...outer part of the retina.  Find the AAV synthetic promoter plasmids! Comparing synthetic promoters performance...  Applications for synthetic promoter AAVs Besides restricting AAV transgene expression to particular cell... A toolkit of synthetic AAV promoters The Roska lab’s synthetic promoter AAVs allows for targeted gene...about using AAV for retinal gene therapy Find more about precise neuronal targeting using AAV technologies...

...blog Viral Vectors 101: AAV Variables That Matter Viral Vectors 101: An Introduction to AAV References...recall, are the outer shell of an AAV vector, which determine the vector’s tropism, including its ability...precious cargo space in AAV vectors. Dual expression systems, which use two or more AAVs to transduce the same...payloads. Since most systemic vectors are primarily derived from AAV9, the vectors have to be administered ... against AAV9-based vectors (and vice versa), enabling repeat dosing (Chen et al., 2023). AAV-BI30, developed...    Figure 1: AAV toolkit for targeted gene delivery to the brain. The AAV (a) capsid, (b) cargo...animal (i.e., 10 and 50 µL of vector at 1 x 1013 vg/mL titer), while AAV-PHP.S is typically used at a ...

...Browse Our Viral Vector Pages Learn About Viral Vector Biosafety Deposit Your Viral Vectors ...specifically AAV, are also being exploited as delivery mechanisms for gene therapy. The tractability of AAV, low...We offer a variety of viral vectors, viral services and even a Viral Vectors eBook to get you started. ... terrains. They are, in fact, a choice delivery vector from basic science research all the way to clinical...of virus to choose from (lentivirus, retrovirus, AAV, etc.). When you’ve decided on an application, our...sequence for degradation. Viral introduction of shRNA plasmids can induce gene-specific silencing temporarily... long-term, and even inducibly depending on the vector and virus. Viral CRISPR-Cas9 tools have been...

...principle of Tetbow In modern neuroscience, plasmid or viral vector-mediated gene delivery is more common than...four Tetbow plasmids (Addgene #104102 - #104105) using in utero electroporation or with AAVs (prepared ...troubleshooting your Tetbow experiment Plasmid concentrations and AAV titer - The XFP genes have to be introduced...highest color variations. Adjust the plasmid concentrations or AAV titer for the best results. The timing...Brainbow Learn about using AAVs for neuronal tracing Visit the viral vectors blog topic page Additional...than conventional transgenic strategies. In such vector-based gene delivery systems, the stochastic expression...sacrifice the animals and fix the samples. When you use AAVs, it typically takes a few weeks to achieve optimal...

...calculator for AAV packaging Select the plasmid molar ratio you would like to use for your AAV packaging ...names of the plasmids you plan to use Enter the base pair size of those plasmids Enter plasmid concentration...Using AAVs AAVs Are Great Tools for Gene Expression in Mammals Resources on Addgene.org Find AAV Packaging...PEI transfections for AAV packaging, considering using this PEI Calculator. AAV packaging typically requires... to making AAV packaging a little easier! Click here to download the PEI calculator for AAV packaging ...ratios below: 1 Transfer Plasmid : 1 pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper: 2 RepCap Select...of each plasmid needed per dish or stack. Calculations are based on using 28 μg of total plasmid (Transfer...

...relevant plasmid depositors and their institutions to distribute viral preps derived from plasmids in the...distributing a small but growing catalog of ready-made AAV and Lentiviral preps. This new Viral Service represents...largest new initiative since we started distributing plasmids in 2004. We’ve already distributed over 500 viral... virus. Addgene is a 501(c)3 nonprofit and our plasmid service was started based on donations. That seed...depositors. Over 3,000 laboratories are sharing plasmids (with 16 labs currently sharing viral preps) through... lab from the Allen Institute have supported the AAV initiative from the beginning and have worked closely...Research Campus have provided valuable insight into AAV production. Finally, we are blessed to have an incredibly...

...Since Addgene began producing AAV vector preparations, we have used quantitative PCR to evaluate the ...powerful tool for quantifying AAV for some time now. When titrating AAV by qPCR, viral DNA is amplified...posts related to viral vector protocols and tips Read all of our viral vectors blog posts Resources on...Learn our protocol for AAV titration by qPCR Browse our collection of viral vector related protocols Learn... is where droplet digital PCR (ddPCR) comes in. AAV titering using quantitative PCR Before we dive into...reactions with known quantities of DNA. Additionally, an AAV reference, a virus with a known titer, can be used...and both results would be valid. This is why the AAV reference is critical. It allows you to determine...

...deliver in low-capacity vectors like AAV. AAV (adeno-associated viral vectors) are commonly used for in..., these vectors have a low genetic capacity of ~4.5 kb. At 4.1 kb, SpCas9 barely fits into AAV when you...previously used SaCas9 (1053 aa/3.2 kb) with AAV vectors for efficient postnatal editing. LbCpf1 and AsCpf1...SpCas9. These nucleases should fit well into AAV vectors, especially given that Cpf1 gRNAs are also shorter... is well suited for genome editing.  Find Cpf1 Plasmids at Addgene   Image attribution: Zetsche...potential to eclipse any of the CRISPR nucleases in AAV-based applications. Get More Cpf1 Background Here...Biotechnol. 2016 Jun 6. PubMed PMID: 27272384 Find plasmids from this publication at Addgene. 2. Kleinstiver...

...an engineered adeno-associated virus (AAV) vector named AAV.CAP-Mac for non-invasive systemic gene delivery...of the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles.  ...all applications. Addgene’s plasmid collection includes over 10,000 plasmids with a FLAG tag (!), but did...from variable region VIII of AAV9-X1.1 to that of AAV1. This viral vector has brain endothelial cell-specific...Importantly, AAV1-X1 is not neutralized by pre-existing antibodies against AAV9-based vectors (and vice ... enhanced brain potency of AAV-PHP.eB.    Figure 4: AAV9-X1 and AAV9-X1.1 efficiently transduce... variants AAV.CAP-Mac for systemic gene transfer in primate brains Endothelial-specific AAV delivery Recombinant...

...of Genome Engineering Using FLEx AAV Vectors? Learn about Viral Vector Elements Read Our Primer on Optogenetics...improvement of optogenetic adeno-associated virus (AAV) vectors. Listed below are a few additional uses for ...In a previous post from our Plasmids 101 series, we learned how the Cre-loxP recombination system can... even in the presence of Cre recombinase. This plasmid now specifically drives expression of mCherry instead...Since most optogenetic reporters are delivered using AAV injection, the majority of the cells surrounding ...2286602. PubMed Central PMCID: PMC3406316. Find FLEx plasmids from these Addgene depositors: Ed Boyden Bernardo... plasmids...

...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...Biology Find plasmids containing these base editors Spatially and temporally regulated AAV-based genome...developed another AAV toolkit - this time using the Staphylococcus aureus Cas9 (SaCas9). This AAV-based genome...Molecular Neuroscience Find the AAV-based SaCas9 genome editing plasmids String assembly gRNA cloning...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...paper in Nature Communications Find the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase...single step gRNA multiplexing system that generates vectors containing high numbers of gRNAs in one overnight...

...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...They generated plasmids tagged with EB3 to mark the growing tips of microtubuli and plasmids tagged with ...and dCas9-VPR targeting plasmids can be found at Addgene. Check out these plasmids for piggyBac gRNA delivery...highlights from recent CRISPR plasmids. To find all of the CRISPR plasmids available from Addgene, head... Find the plasmids at Addgene The MoClo CRISPR/Cas Toolkit for plants includes 95 plasmids consisting...and dicots. Find the plasmids at Addgene! CRISPR RNP electroporation and AAV donor infection (CRISPR-READI...

...Production at Addgene Viral Vectors 101: What is a Virus? Viral Vectors 101: Viral Vector Elements ...adenovirus, or AAV. Then, your desired DNA to be integrated can be cloned into the vector that you have...non-integrating (adenovirus and AAV) but can still express episomally long-term (AAV). If you perform a lipid-based...nucleic acid cargo of your choice. First, a viral vector is selected to perform the transduction with: either...produced, and viral particles are packaged, when the vector is expressed. Once the design and construction ... it’s time to make virus! The constructed viral vector containing the cargo of interest is then typically...typically transfected along with viral packaging plasmids into a viral production cell line, usually HEK-293...

...elements are inserted into an AAV transfer vector and used to generate AAV particles. Transient or stable... in a single lentiviral transfer vector or separate transfer vectors. May contain reporter gene (e.g. ...non-dividing cells. AAV is least toxic method for in vivo viral delivery. Cas9 mRNA and gRNA Plasmids containing...add a tag, you will also need to deliver a donor plasmid or single-stranded DNA oligonucleotide, so you ...Components of System Application Mammalian expression vector Promoter driving Cas9 expression can be constitutive...generate stable cell lines. Packaging and Envelope plasmids provide the necessary components to make lentiviral...for conducting genome-wide screens using CRISPR. AAV transduction Only compatible with SaCas9 (packaging...