.... CasPEDIA Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes [Preprint]. Biochemistry.... (2019). Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. ...resource that provides summary information on Class 2 Cas enzymes. The information is organized in wiki ...this content and much, much more.     Fig. 2 – List of resources and properties for a Cas entry.... Nature Reviews Microbiology, 18(2), 67–83. https://doi.org/10.1038/s41579-019-0299-x Resources on Addgene.org...established uses, and intended applications. Within CasPEDIA, there is a classification system, CasID, to help...multiplex ability Experimental guidelines and applications Protein structure and properties Links to ...

...Containing Multiple Introns.” Plant Communications, vol. 2, no. 2, Elsevier BV, Mar. 2021, p. 100135. ...Constructs.” PLoS ONE, edited by Jean Peccoud, vol. 6, no. 2, Public Library of Science (PLoS), 18 Feb. 2011, p... vol. 5, no. 10, American Chemical Society (ACS), 2 May 2016, pp. 1059–69. Crossref, https://doi.org/10.1021...of Plant Genomes.” The Plant Genome, vol. 16, no. 2, Wiley, 9 Mar. 2023. Crossref, https://doi.org/10.1002...Crossref, https://doi.org/10.1186/s12870-020-02388-2. Stuttmann, Johannes, et al. “Highly Efficient Multiplex...Crossref, https://doi.org/10.1038/s41598-019-46171-2. Resources on Addgene.org Modular Cloning (MoClo...and applications.    Plant applications We outlined many synthetic biology MoClo applications, but...

...alkaline lysis plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid DNA This kit-free...protocol: 1) it solubilizes both DNA and agarose, and 2) it helps DNA stick to the glass. After binding DNA... had a similar level of sensitivity. Figure 2: Steps to purify DNA using a cellulose dipstick. Image...Academy of Sciences of the United States of America, 76 2, 615-9 . https://doi.org/10.1073/pnas.76.2.615  Zhou... DNA purification without a kit as well as one way to reuse silica columns from DNA purification kits....consider some ways to purify DNA without a kit. DNA purification kits have advantages: they are convenient and...because you’ve run out of columns. Kit-less DNA purification methods avoid many of the drawbacks of kits ...

...SARS-CoV-2 spike pseudotyping, visit the SARS-CoV-2 Pseudotyped Virus collection page. SARS-CoV-2 detection...of SARS-CoV-2 entry and become upregulated during viral infection.  Expression of SARS-CoV-2 viral proteins... expression of cathepsin S, L, and B. This became a great starting point for SARS-CoV-2 researchers to...labs in over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman... protein, variants, and pseudotyping The SARS-CoV-2 virus depends on its spike protein to bind ACE2, the...of infection and how to interfere with it in a BSL-2 lab instead of a BSL-3 lab. Jesse Bloom’s lab published...reagents for pseudotyping lentivirus with the SARS-CoV-2 spike protein. They deposited the plasmids used for...

... and then elute YFP.  Option #2: Opto-Nanobodies (OptoNBs) purification You skip adding a tag to YFP and...choose?  Option #2 may sound like fiction, but light-controlled protein purification is now a reality ...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...#1: Affinity tag purification You tag YFP and use an affinity column for purification. After binding YFP...  Light controlled protein purification with OptoNBs The first application for OptoNBs takes us in vitro...light-controlled affinity purification of unmodified proteins. Affinity purification methods typically require...It’s time to choose your own protein purification adventure. You want to purify your favorite protein...

...tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for ...endeavor. Case study 2: Plasmids Another example of reagents that have broad applications but have proven ...used in a publication by Xiuyuan Ou, et al. that characterized the spike (S) protein of SARS-CoV-2 (Ou et.... This publication then resulted in the development of a new plasmid, pCMV14-3X-Flag-SARS-CoV-2 S, which...fast access to the tools required to study SARS-CoV-2, but with many academic research labs closed during...biobanks, are crucial for the advancement of SARS-CoV-2 research because they provide rapid access to validated...Many of the reagents used to investigate SARS-CoV-2 were developed for broad use and are now important...

... professionally, and 2) I needed to start using Twitter. Sharing Chemistry Cat memes use to be how ... writing for the Addgene blog, sharing Chemistry Cat memes was how I used social media as a scientist....but my consistent writing for the Addgene blog (~1-2 blog posts per month) and use of Twitter and LinkedIn...PhD 👩🏾‍🔬💜 (@ThePurplePage) February 13, 2014 2. "The Scientist's Top 10 Guide to Twitter" blog post...you social media can positively impact your career, 2) given you tips for using various social media platforms...remotely as an Addgene Science Communication Intern. Developing science communication skills, while also learning... on a panel about “Social Media for Science Communication” at the American Society for Cell Biology (ASCB...

...editing (Fig. 2 and 3) (5, 3). Benefits of truncated gRNAs Importantly, the use of truncated guides has ...shown to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative...PMID: 24076762. PubMed Central PMCID: PMC3844869. 2. Chari, Raj, et al. "Unraveling CRISPR-Cas9 genome...protospacer, in the genome. Native Cas9 has two catalytic domains, each of which cleaves one strand of DNA... addition to its utility in making targeted modifications to DNA, Cas9 can be reprogrammed to serve as...as a regulator of gene expression. Its catalytic domains can be mutated to inactivate the protein’s nucleolytic...target sites. Regulating gene expression with truncated gRNAs An alternative approach is to modulate Cas9...

...discussed them in meetings and 2) creating a pictorial guide for our engineers (Fig. 2). Creating this guide was...in improving cross team communication has helped Addgene move from completing 2-3 large projects a year.... I needed to a) figure out the sources of miscommunication and b) find practical solutions.  My PhD ... for this task. Good scientists learn how to communicate their work differently based on their audience... family is going to vary widely. The way you communicate your science at a poster is going to vary depending...two main problems with Addgene’s cross team communication. Although these real world examples did not ... for the container to be used in cross-team communication A list of “AKA” names, or jargon terms, for ...

...pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 N protein  pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro...SARS-CoV-2 M protein pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 ORF3a... SARS-CoV-2 protein expression The Krogan lab quickly shared plasmids expressing SARS-CoV-2 proteins at...pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro) and NSP2 (pLVX-EF1alpha-SARS-CoV-2-nsp2-2xStrep-IRES-Puro...months: Lentiviral preps for expressing SARS-CoV-2 proteins ChR2 for targeted expression DREADDs GFP ...Serotype Name 83899 AAVrg pAAV-mDlx-GCaMP6f-Fishell-2  Additional resources on the Addgene blog Read...availability date or to be notified when a particular catalog item is available for order please email [email protected]...

...neoblast cells and the gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating... characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a specific mRNA sequence...223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver...DE3. For Phagocata morgani and Phagocata gracillis, the process is a little more complicated. These strains...Girardia sp., Dugesia dorotocephala, Phagocata morgani, and Phagocata gracillis. There are 11 different ... coli. Thus, to get the dsRNA into Phagocata morgani and Phagocata gracillis, one has to purify the dsRNA... in situ hybridization experiments. A recent publication [1] from the Sánchez lab describes the plasmids...

....9 (parental) 46 +/- 0.7% AAV.PHP.S 82 +/- 2% Table 2: Transduction efficiency of AAV.9 versus AAV.PHP.S...figure 2) to allow for efficient IV delivery of virus to the CNS or PNS (figure32, tables 1 & 2). These...because: 1) their genomes are easy to manipulate, 2) they have long-term expression; and 3) they have ... On average, AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains ...stained for a neuronal marker). Check out tables 1 and 2 below for a summary of the quantified transduction...cells are labeled with their own unique color; and 2) low levels of labeling so that a cell’s projections...maximize the number of cells that are labeled. Part 2: An AAV expressing the inducer (i.e. tet-off transactivator...

... to isothermal amplification by primers that target the E and the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA...case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification was able to detect as low ... State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...Rapid detection of 2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-... method has been optimized to detect the SARS-CoV-2 virus directly using saliva without having to perform...and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies/microliter of RNA targets...easy-to-deploy, protocol for Cas13-based detection of SARS-CoV-2 genetic material (CREST) Sensitivity: 10 copies of...

...the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two ...Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello JA, Lichtman JW. Optical...enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict excitation (and bleaching...can be seen exiting through the tissue. Therefore, 2-photon allows for deeper imaging into thick tissues...imaging objective that collects fluorescence. Like 2-photon microscopy, only a single focal plane of the...relying on raster scanning as is done in confocal or 2-photon microscopes. Total Internal Reflection Microscopy...

...Illustration of SARS-CoV-2 from Philippa Steinberg for the IGI.   What SARS-CoV-2 plasmids from Ginkgo ...intended for the high level expression and purification of SARS-CoV-2 proteins for downstream experiments and...and codon optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned into a CMV...Bioworks to synthesize and share over 1,850 SARS-CoV-2 plasmids to help academic and industrial labs accelerate...request? Currently, plasmids containing all SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...bacterial expression systems, Ginkgo synthesized SARS-CoV-2 expression plasmids in a high copy backbone with a...first deposited set of plasmids containing SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...

...comprehensive SARS-CoV-2 virus-human interaction network in which we expressed 26 of the 29 SARS-CoV-2 viral proteins...SARS-CoV-2 viral proteins except for Nsp3 and Nsp16, and includes an additional catalytically dead version...all became acutely aware of the threat of SARS-CoV-2. Like many in the science community, we’ve been motivated...identifying these potential human helpers of SARS-CoV-2 (especially the ones that are druggable) is a key ...just at the beginning of understanding how SARS-CoV-2 interacts with human proteins. For example, we used... joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in various ways by the...is going on there!).  Find the Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC...

... non-physiological, and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several...Biol, 2003. 163(2): p. 257-69. PubMed PMID: 14581454. PubMed Central PMCID: PMC2173526. 2. Zacharias, D.... nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]....form dimers in a cell. The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined above, you might...organized smooth endoplasmic reticulum (OSER) whorls. 2)A dimerizing protein of interest fused to a dimerizing... designed to detect resulting in false positives [2, 4]. Solutions to the polymer problem There are some...Citrine) can be monomerized with an A206K mutation [2]. Addgene has many of these constructs and they are...

...status of MEK1/2 was tracked at 5 minute intervals via Western blot. Phosphorylation of MEK1/2 peaks at just... phosphorylated MEK1/2 levels, suggesting PP1/PP2A is responsible for this MEK1/2 negative feedback. ...PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot, Sergi, Jacob J. Hughey...precisely control the timing of kinase activity. 2. Studying negative feedback loops of signaling pathways... studied the negative feedback on endogenous MEK1/2 phosphorylation by creating a short pulse of psRaf1...activity. They did this by exposing psRaf1 to a short 2 minute pulse of 500 nm light (turn on psRaf1) followed...members of their pathways, but where the kinases are located, their signal strength, and how long their signals...

...increase in virus. Purification (2 days) - Purification is required if rAdV is to be used in vivo. The standard...incubation as it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more... this “cytopathic effect.” Amplification can be repeated multiple times (2-4 rounds) at increasing scale...scale over the course of 1-2 weeks. Each round of amplification should result in a 10-100-fold increase...creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...sequenced to confirm its integrity. Initial production (2-3 weeks) - Here you’ll produce the primary recombinant...

...cancer. These viruses need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.... BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such...diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) and gamma...recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus, many organizations...sprayed into the air. For lentivirus and other BSL-2 materials, you’ll need to use aerosol-tight caps, ...percentage of the decontaminate to be used for BSL-2 waste, and of course, if you are working with a substance...host genome, the virus is replication incompetent, meaning it can’t replicate and continue the infection...