...journal club discussion on Kevin Esvelt’s CRISPR/Cas9 gene drive paper was very popular. People kept asking... at Addgene.org Order Plasmids from Addgene! Find Free and Open Educational Resources at Addgene! ...BosLab. Once he optimizes plant cell culture and transgenesis he wants to share his knowledge with the community...microbes. We’ve ordered a few yeast plasmids from Addgene and are eager to do our first yeast transformations... @ZebraElement     Additional Resources on the Addgene Blog Experimenting in an Open Source Lab: from...

... Intersectin, Cdc42, Raf1, Mek1, Mek2, Cdk5, and Cas9. Xin has demonstrated the utility of these FLiPs...Fatima's work and keep checking back in with the Addgene blog for future award opportunities. Xin Zhou:...when exposed to ultraviolet light. To do so, she generated fusion proteins in which Dronpa domains flanked...Proteins, or FLiPs.  Xin later optimized these first generation FLiPs by mutating Dronpa to lower its aggregation...Chemical Biology! Additional Resources on the Addgene Blog Learn More about Photosactivatable Fluorescent...Fluorescent Protein for Your Experiments Resources on Addgene.org Find Fluorescent Protein Biosensors Browse Plasmids...

... transcription of a target gene, it therefore determines the timing of gene expression and largely defines...activating transcription. Addgene depositors have developed heat shock-inducible Cre and Cas9 for easy genome engineering...synthesis and allowing reporter gene transcription to take place. Addgene also has yellow flame light-regulated...the promoter and ceases repression of the target gene. A simplified lac inducible system is found in many...shock-inducible Hsp70 or Hsp90-derived promoters, in which a gene of choice is only expressed following exposure to...inducible promoters Light is another way to activate gene expression, and two-component systems used in synthetic...promoter pR, preventing expression of a reporter gene. When light is present, YFI is inactive, preventing...

...all known genes. This is a perfect example of an unbiased, reverse screen – all known genes are assayed...of knocking out the genes of interest (or the whole genome), they knock down gene expression temporarily...Sabatini, D.M., Lander, E.S. Genetic screens in human cells using the CRISPR/Cas9 system. Science, 343(6166... a biological process. Players can be proteins, genes, enzymes, drugs, or anything else that plays a role...where there is removal of known factors (such as a genes) and the loss of those factors is assessed. The ...especially useful if you are interested in studying genes whose knockout would be lethal to cells and thus...The two most common methods of manipulation are genetic knockout mutations and treatment with small molecules...

...used with many types of proteins, including Cas9 and dCas9-transcription factors, for which low expression...plasmids from this publication at Addgene. Multichromatic control of gene expression in Escherichia coli...optimized by Tabor’s lab, making light-controlled gene expression in E. coli easier and more robust. Synthetic...used to engineer new pathways with sophisticated gene expression controls. Most cell types do not respond... can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive two-component... was only about 10-fold, precluding the study of gene expression at very high or very low levels. Schmidl...researchers to better fine-tune the level of output gene expression based on the input light intensity. In...

...ampicillin resistance gene, you’ll find the full name of the gene, the name of its gene product, and a description...features, such as Cas9, weren’t detected by our in-house software. In fact, SnapGene’s feature library ... plasmid features such as antibiotic resistance genes, selectable markers, promoters, origins, tags, and... Overview of SnapGene powered plasmid maps at Addgene  Three factors make our SnapGene powered plasmid...powered by GSL Biotech's SnapGene Server Software. With the backing of SnapGene’s sequence viewer software...the features you need quickly. SnapGene’s Feature Library - SnapGene’s software seamlessly identifies ...scientists) for suggesting these changes, and the SnapGene and Addgene software development teams for working tirelessly...

...from mice that constitutively express Cas9 and screen for genes that affect Th2 differentiation and activation... popular tools for genetic screens, enabling genome-wide interrogation of the genes required for cellular...inserting the NanoLuc luciferase downstream of the apoB gene. Using this reporter, the researchers explore many...-LPs and accelerate the discovery of additional genes and small-molecule probes to aid in the understanding...transduction, now enabling forward genetic screens in cells with few genetic tools. Find the mouse CRISPR ...plasmids at Addgene. Liu et al., Nature Communications, 2019. PubMed PMID: 31451697. Genetically encoded ...responsible for a phenotype can be identified by next-generation sequencing of the genomic DNA. However, some ...

...changes and require updating or retiring. CRISPR/Cas9, for example, has gone through rapid change in the...everything else we do! The goal of Addgene’s educational content Addgene truly believes that sharing speeds...you ever wondered how we choose topics for the Addgene blog? Or why we make certain videos? In this post...You probably won’t be surprised to learn that Addgene’s mission statement extends beyond plasmids, AAVs...topics. What better space to do that than in the Addgene blog?  Fig. 1: One of our favorite content...content collections is our Help Center, help.addgene.org, where you can easily search articles answering ...sure things are clear, accurate, and in line with Addgene style.        Fig. 2: Reference image (...

... delivery of CRISPR/Cas9 Tips for a first time AAV user Resources on Addgene.org Find Adenovirus Plasmids...Technique Q1: Is there any way to deliver a long gene (>4.5 kB) by AAV? A: One potential strategy is to...applications in diseases which may be linked to epigenetic changes, such as multiple sclerosis?  A: Basic...an exciting area for future AAV application to epigenetic editing. This area is discussed in our recent...excellent questions.    Additional Resources on the Addgene Blog Using virus in your research - a primer for...

...eNLs as good reporter genes for endogenous protein fusions created using CRISPR/Cas9. eNLs offer high luminescent...plasmids from this paper at Addgene. Additional Resources on the Addgene Blog Learn about Photoactivatable...proteins. To make luminescent imaging a reality, Addgene depositor Takeharu Nagai and colleagues at Osaka...nano-lanterns for use in various applications, including optogenetics, biosensors, and fusion proteins. Solving the...yield, and when transiently expressed in cells, generates much less signal than comparable fluorescent proteins... approach is especially suited to complement optogenetic tools, where light used for excitation can cause...cause unintended activation of the optogenetic system. Biosensors based on Nano-lantern technology are...

...situ is the knock-in of fluorescent tags into the gene locus of interest. An advantage of this alternative...translation in living single cells. However, before CRISPR-Cas9 these techniques were quite tedious and, even here...studied a synaptic protein that is linked to neurodegeneration. The level of this protein is decreased in...PMCID: PMC4121734. Additional Resources on the Addgene Blog Fluorescent microscopy techniques - Which... tagging strategies Additional Resources on Addgene.org Fluorescent protein plasmids Use FRET to study...protein-protein interactions Deposit plasmids with Addgene! ...

...label cells. If GFP could be co-opted to control gene expression, he could then selectively manipulate...used LexA and rTetR systems such that they activate gene expression from different promoters. Moving to an.... GFP expression robustly activated the reporter gene TdTomato, whose expression was absent without electroporated...sites. Placing a lox-STOP-lox cassette upstream of a gene of interest blocks transcription when Cre is absent...present, it removes the STOP cassette, activating gene expression. Although Cre isn’t a modular protein...versions of non-modular proteins like Cre. Could Cas9-DOG be developed soon to allow genome modification...fluorescent proteins as scaffolds for cell-specific gene manipulation.” Cell 154(4) (2013): 928-939. PubMed...

... containing structural genes and a transfer plasmid carrying the transfer gene. By dividing the viral ...while a single 72h harvest is best for larger genes such as Cas9. See figure 1 for an example of how changing...Talking 'bout my (lentiviral) generation The first step in delivering genes via lentivirus is deciding what... While lentiviral vectors are popular gene delivery tools, producing lentivirus, can pose certain challenges...split the viral genome by removing the viral Rev gene from the packaging plasmid and incorporating it ... the structural elements, envelope, and transfer gene are transfected into a packaging cell line, usually...sufficient titer; this is often the case with large genes. This is due to inefficient packaging of large inserts...

...., 2018). Hybridomas can lose gene expression of the mAb-encoding genes or fail to revive after cryopreservation...monoclonal antibodies that are generated in vitro using synthetic genes that are typically expressed from... integrated sequence in a stable cell line. The genes encode the heavy chain and light chain for the antibody...Figure 2: (Left) The light chain and heavy chain genes of the antibody are encoded in a plasmid. (Right...better and better at identifying and cloning the genes for antibody expression. If starting from a protein...sequences of the antibodies and then synthesize the genes that encode those amino acids (Tran et al., 2016...community has created hundreds of useful variants of Cas9. By providing access to the plasmids encoding antibodies...

... at Addgene 2. Green, Alexander A., et al. "Toehold switches: de-novo-designed regulators of gene expression...was an in vitro paper-based platform that allowed gene circuits to be deployed out of the laboratory in...combined technologies, we built paper-based synthetic gene networks for the detection of antibiotic resistance...-of-concept work was exciting because it enabled gene circuit-based sensors to be used outside of the ...amplification, new toehold switch designs, CRISPR/Cas9-mediated genotyping, as well as our electronic reader... 3. Pardee, Keith, et al. "Paper-based synthetic gene networks." Cell 159.4 (2014): 940-954. PubMed PMID...Sensor Constructs at Addgene As we report, we have embedded this next generation of toehold switch-based...

...sequence to confirm backbone elements.   Confirming the gene/insert by aligning to NCBI entry or using BLAST....available reference sequence or plasmids containing genes with many modifications. We look for point mutations...amino acid. We also confirm that the species of the gene matches the data associated with the plasmid.   ...Viral Preparations from Viral DNA Extracts. Human Gene Therapy 31:664–678 . https://doi.org/10.1089/hum..., Sanjana NE, Zhang F (2017) Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening...for all incoming plasmids was a big change for Addgene - we had used Sanger sequencing for quality control...control for over ten years. Now, thanks to a generous donation by the Hirsh family, we are able to take...

...et al., Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in... AdVs are stripped of regulatory genes E1 and E3. Without these genes, AdVs cannot replicate on their ...et al. "Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in...applications and gene therapy: they can be produced at high titers, can accommodate large transgenes, transduce...recombination-competent adenovirus (RCA) can be generated if the E1 gene from the packaging cell line is transferred...for the generation of RCA and reduced immunogenicity. In addition to E1/E3, non-structural genes E2 and...proteins in trans. The Gutless AdV have generated high interest for gene therapy due their increased cloning...

...variations in gene expression, they found that for approximately 40% of the identified genes, the genes were not...explanation. Next, they hypothesized that differences in gene expression could explain why some ancestry groups...groups were differentially dependent on certain genes. This didn’t prove to be correct, either. However, ...sequences for sgRNAs that target ancestry-associated genes.   What does the reference genome actually reference... the guide from binding to the genome or prevent Cas9 from cutting, both of which can introduce significant...cases, this may lead researchers to deprioritize genes that could be good targets for therapeutic development...cells’ germline genetic variations. They then wanted to identify cancer-associated genetic dependencies ...

...overexpress genes of interest; more recently we have also been using Addgene CRISPR vectors to generate cell ...cell lines with knockouts of specific genes. Many years ago, people in the laboratory became frustrated with...cDNA is co-transfected at a favorable ratio to the gene of interest (or, if your protein is fused with a...well plate for further analysis. To assess CRISPR/Cas9 knockout efficacy, we have used both Western blotting...with different levels of expression of our gene of interest. Advantages of the agarose cloning method ...figure 1.   Find Additional Useful Protocols on Addgene's Protocol Pages Agarose cloning method procedure...-well cell plate to a 96-well assay plate. We generally freeze at least 2 ampules each of 4 clones with...

...   For CRISPR libraries, Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening...Libraries Resources on Addgene.org Addgene's Guide to Plasmid Pooled Libraries  Addgene's Pooled Library Collection...chemical transformations are generally cheaper and easier. One library that Addgene distributes with a chemical...contact us at deposit@addgene.org. Happy amplifying! More resources on the Addgene blog Genome-Wide Screening...If you’re depositing a pooled library with Addgene, you may be surprised to learn that we ask for an ...recombination and loss of plasmid diversity, so Addgene strives to distribute aliquots of the original ...requesting scientist receives a pooled library from Addgene, they’ll need to amplify the sample for further...