...expression vectors with various promoters suitable for in vivo expression, and/or produce vectors to make...of recombinant DNA technology has allowed molecular biologists to fuse fragments of DNA together in a ...prototrophic strains. The vectors were based on the original EasyClone vectors (Jensen et al 2013) allow...CDC and cloned into a third generation lentiviral transfer vector. Each gene has been FLAG-tagged for convenient...donor vectors containing mAID and either Neo or Hygro resistance markers with CRISPR/Cas vectors targeting...the authors packaged it into lentivirus and used the resulting lentiviral library to generate a population...simultaneously incorporate multiple single-strand DNA (ssDNA) oligonucleotides (oligos), and thereby rapidly...

...derivatives. Unmodified Cas9 causes double stranded DNA breaks that commonly result in a complete loss of...Brunello CRISPRko sgRNA library as pooled library or lentiviral prep! Dolcetto: human CRISPRi sgRNA library While...These mutations result in an inactive RNA-guided DNA binding protein (dCas9). dCas9 is then tethered to...the Dolcetto library! Now also available as a lentiviral prep. Calabrese: human CRISPRa sgRNA library ...tracrRNA was combined with the sgRNA expression vector allowing the screen to be conducted with only two...function. Find the Calabrese pooled library and lentiviral prep here! Applications for the Root and Doench...viability genes as they are subject to an increase in dsDNA breaks that can cause cell death. Thus, CRISPR inhibition...

.... At the time, adenoviral vectors were the most commonly used viral vectors for gene delivery, and they... better vector for ocular gene therapy: Adeno-Associated Virus (AAV) While other viral vectors such as...gene can be targeted to RPE cells using a lentiviral vector and a clinical trial is ongoing. Moving forward...AdV) and lentivirus (LV) are known to transduce retinal cells, AAV quickly became the vector of choice...technician in a lab using adeno-associated viral vectors (AAVs) to treat an inherited retinal degenerative...tolerate the presence of antigens (such as viral vectors) without triggering a destructive inflammatory ...retina but only transiently. However new viral vectors capable of providing long-term gene expression ...

...expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast (52962-LV, Figure 1). Some...primers flanking my edits in genomic DNA and doing PCR on genomic DNA. The surveyor assay itself was relatively...that Surveyor nuclease cleaves double stranded DNA (dsDNA) at basepair mismatches. To use this tool to ... denature and renature the dsDNA. Upon renaturation, you will generate DNA duplexes with one strand from...performing and analyzing a gene edit using Addgene’s lentiviral CRISPR tools. Schematic overview of CRISPR...gRNA-transduced Cas9-A549 cells are used as a control. DNA was analyzed by PAGE. The basis of the Surveyor...these mismatches are indicated by the presence of DNA cleavage products. The appearance of the 428 bp band...

...producing lentiviral and MuLV retroviral particles. Use in conjunction with a packaging vector such as ...as pCMV-dR8.2 dvpr (lentiviral) or pUMVC (MuLV retroviral). Bob Weinberg pMD2.G VSV-G envelope expressing...paper for more information Miguel Sena-Esteves pcDNA-SADB19L Contains rabies viral RNA polymerase; ...

...roughly half the packaging limits of lentiviral and adenoviral vectors. While many transgene will fit within... overviews of three split vector strategies plus one additional split vector strategy for increasing the...homology directed repairs (HDR). Split AAV vector approaches Split vector approaches increase the size of the...copies of both vectors. Examples of use: Halbert et al., 2002 used an overlapping vector with 440 or 1000bp... hybrid vector, as measured by β-galactosidase activity, was 81% of that from a single vector, but significantly...ideal split vector system. Overall the efficiency of transgene expression from split vectors has been inconsistent..., Duan D (2008) A Hybrid Vector System Expands Adeno-associated Viral Vector Packaging Capacity in a Transgene-independent...

...known or suspected to be involved in DNA damage response and DNA repair.  A new CRISPR pooled library ...only required three components: a master mix, the DNA of interest, and cell extract.    Graphical...They allow single-nucleotide modifications to edit DNA without requiring a double-stranded break. The Xiao... a) Cells are transfected with three different vectors and flow cytometry is used to sort populations ... 10 guide RNAs per gene. The lab modified the lentiviral backbone to encode the CD90.1 selection marker...

...Baradaran’s expertise in viral safety, gene therapy vector development, vaccine development, and assay technologies...she says. In 11th grade she heard a talk about Z DNA that inspired her to major in molecular biology in...Harvard Medical School, he developed the first lentiviral microRNA-based system for polymerase II-regulated...Melina Fan and former Associate Director of Viral Vectors Karen Guerin for the journal Molecular Therapy....

...protein. Mammalian vectors One of our most popular mammalian expression vectors, c-Flag pcDNA3, comes from the...will need accessory packaging vectors to generate the virus. Bacterial vectors This system is great for overexpressing...cases bacterial expression vectors are inducible (e.g. pBAD LIC cloning vector (8A)) and have an epitope...folding assays. Yeast vectors There are several types of yeast expression vectors. Consider your applications...shuttle vectors. You may prefer the more traditional approach of an autonomously replicating vector that ...conduct your custom experiment. Fly vectors The vast majority of fly vectors are based on p-element, random... to reporter vectors (e.g. pBV-Luc) and gene silencing (e.g. pLKO.1 - TRC cloning vector). Check out our...

...pandemic, the Krogan lab at UCSF generated lentiviral vectors expressing SARS-CoV-2 open reading frames...which all but one are cloned into pLVX vectors (for lentiviral packaging) and contain an IRES-Puro marker...(2020) Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization...published a protocol and reagents for pseudotyping lentivirus with the SARS-CoV-2 spike protein. They deposited...qPCR and antigen tests.  Labs including Jennifer Doudna’s lab and Feng Zheng’s lab had already developed...

...Addgene.org Browse Our Viral Vector Plasmids Read Our Lentiviral Vector Guide Read Our AAV Guide  ...of producing infectious virus (similar to lentiviral vectors) because they do not encode all of the necessary...on the Addgene Blog Read Other Posts on Viral Vectors Check out Our Scientist's Guide to Ebola Contribute... viral vector...of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication...

...) ChiRP-seq RNA Hi-C DNA (genome-to-genome interactions) DNase-seq DNA (regions that are accessible... regions of DNA that demonstrate “spooky action at a distance.” Through the marvel of DNA compaction, ...details on DNA compaction and the structural proteins that make it possible, see the appendix DNA is a 3D...millions of embryonic stem cells (ESCs) with a lentiviral vector, an attenuated retrovirus in the same family...See below for further details. DNA is a 3D Fractal Figure 2: DNA compaction. Graphics courtesy ...For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning of the third chromosome, ten...coordinates for genetic elements like protein, RNA, and DNA (Table 1). As with GPS in the real world, coordinates...

...macromolecules used in scientific research such as DNA or protein. It comes as no surprise then that stability...Base modifications What is more stable than RNA? DNA! One way to make your gRNA more efficient on a budget...ribonucleotides for deoxynucleotides. A partially DNA gRNA is surprisingly well-tolerated by the Cas9 protein....org  View Addgene's collection of empty gRNA vectors View Addgene's CRISPR resources Request ready-to-use...-to-use viral preps of select CRISPR lentiviral plasmids Additional resources on the Addgene Blog  How...

...  The RNA polymerase(s) RNA is transcribed from DNA using an RNA polymerase (RNAP). In bacteria this ... as RNAP II promoters is commonly employed in lentiviral and retroviral constructs and we will discuss...discuss these in a future post on viral vector parts. Promoter specificity Aside from choosing a promoter based... Can be induced by IPTG. Commonly found in pET vectors. Very tightly regulated by the lac operators. Good...-inducer fucose Weaker. Commonly found in pBAD vectors. Good for rapid regulation and low basal expression...Browse All Plasmids 101 Posts Learn about Mammalian Vectors Read about Reporter Gene like Luciferase and GFP...

...connectors, cDNAs, and more to find the perfect combination. The EcoFlex Kit has a wide array of DNA parts ...Modular cloning is a popular DNA assembly tool used to build single- and multi-gene constructs for a ...background MoClo is a clever way to assemble several DNA parts in a specific orientation using Type IIS restriction...kit has a focus on infrastructure with shuttle vectors, several delivery systems which can be engineered...engineered into the TUs, and even viral gene silencing vectors. The Plant Parts III kit has a focus on transformation...Fragmid system was used to assess how different lentiviral plasmid backbone elements impact viral titer ...assemblies or that compare the effects of different DNA elements on the behavior of an expression construct...

...via a lentiviral vector to cell lines that either stably expressed Cas9 or that had been lentiviral infected... non-targeting guides. Toxicity is likely due to DNA damage and its subsequent repair. Additionally, the...fail to account for the effects of Cas9-induced dsDNA breaks. Are off-target cutting patterns in genome-wide...function, i.e. sites that lack open chromatin marks, DNase hypersensitivity, or are in an enhancer, transcription...control for the effects of guide expression and dsDNA breaks. Figure 2: Non-targeting vs. safe-targeting...for the inherent effects, or background noise, of dsDNA breaks caused by CRISPR. See supplemental figure...cheat sheet Read our Quick Guide to All Things Lentivirus Resources on Addgene.org Check out this guide...

...intermediary DNA replication step between DNA and mRNA. DNA genome replication can involve an RNA or dsDNA intermediary...Viral Vectors 101: Viral Applications Viral Vectors 101: Viruses as Biological Tools Viral Vectors 101:...create far safer viral vectors that can be used in BSL-1 or BSL-2 labs. Viral vectors are viruses that have...increase the viral vector’s carrying capacity. In order to allow the viral vectors to reproduce when experimentally...expressed via cells cultured with the vectors. This allows the viral vectors to reproduce only in experimentally...of Viral Vectors 101 posts about each type, so be sure to check our blog, or our Viral Vectors 101 eBook...Louten, 2016 Viruses with DNA genomes can either follow central dogma (gDNA to RNA to proteins) or require...

...integrase-defective lentiviral vectors - which rely on insertion of DNA by the NHEJ pathway during DNA repair of...either introduces a ssDNA nick, a dsDNA break, or is engineered not to cleave DNA and instead activates...an experiment. For CRISPR work, this often means DNA cleavage at unanticipated sites. While off-target...genomic locus must often be proximal to a PAM site for DNA cutting to occur. The RNA sequence and PAM site confer...more. While such software typically only considers DNA sequence and doesn’t take into account chromatin ...only reduce off targets where nicking/cleaving of DNA occurs, not for deadCas activator/repressor experiments...generated near each other, this will result in a DNA double strand break (DSB), the event required for...

...in a mammalian expression vector (pcDNA3) and a bacterial expression vector (pRSETB). Fabritius A, et...like APOBEC. To achieve this they created a lentiviral vector system that monitors both APOBEC- and Cas9...

...fragments of DNA from the helper plasmids or the cell genome can be packaged inside the vectors during production...sequence all the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from... common to find DNA from the packaging cell genome, bacterial genomes, cloning vectors, and helper plasmids...mixed another AAV vector with AAV-68544 to validate our method. We then extracted the DNA and processed the...used to identify DNA contaminants in AAV preparations and provide information on these DNA species with exquisite... (2015) Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus...blog Check out our Viral Vectors Featured Topic Page Subscribe to Viral Vectors Blog Posts Learn How We...