...activate essentially any gene,” she says. The technical leap from mutating and deactivating a gene or ... and it consists of three components: 1. A nucleolytically inactive Cas9-VP64 fusion; 2. A sgRNA incorporating...distinct effector domains that work together synergistically, modeled after natural transcription activation...demonstrated in a paper in Cell that they could typically achieve 90-99% knockdown with minimal off-target...more about the rules.” He said that there is no theoretical limit on the number of genes that can be manipulated...

...improves upon them. Methods for studying autophagy Biochemical assays Long-lived protein degradation: This...radioactivity in the culture supernatant (this is indicative of protein degradation). To control for protein...’s slow and only measures bulk autophagy. Morphological assays Electron microscopy: Electron microscopy...studying autophagy. This method relies on the identification of autophagic structures based on morphology...available from Addgene. References 1. Rosado, C., Mijaljica, D., Hatzinisiriou, I., Prescott, M., & Devenish...

...time-consuming aspects of traditional cloning is the identification of clones that actually contain your insert...enzyme-mediated disruption of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful...ccdB and therefore would die. This procedure dramatically reduces the number of clones that do not contain...which can then be used for downstream cloning applications. While these two strains ultimately perform ...and ccdB Survival strains have been developed specifically with ccdB-containing vectors in mind, any plasmid...

... year, narrow by article type (review, patent, clinical study, research article etc). There are also numerous...looking for a particular protocol or are trying to replicate an experiment, the methods section probably won...conclusions. In any case, the reader should give a critical eye to all results in the relation to how they...is science’s way of validating work prior to publication, it doesn’t mean that there are no mistakes or... biochemistry instructor in the Department of Biological Sciences at Louisiana State University. She is...

...including RNPs for indel formation via NHEJ, precise modification with Homology Directed Repair (HDR), CRISPR ...potential to be used for other types of CRISPR applications. Let’s take a look at the key components of ... which means they are non-infectious and non-replicative. Nanoblades are produced by transfecting the ...cytoplasm or pronucleus of the zygote which is both technically challenging and can negatively impact zygote ...with Cas9-sgRNA ribonucleoproteins." Nature communications 10.1 (2019): 45. PubMed PMID: 30604748. PubMed...

...when it comes to targeting precision and practical application. If the CRISPR/Cas9 system is not effective...but directly incorporating very long ncRNAs is impractical. Instead, tags or stop elements can be inserted...can cleave single-stranded RNA using gRNAs to specifically edit or degrade targeted lncRNAs. The Cas14 ...lncRNAs with high specificity, particularly in applications requiring smaller delivery systems (Zibitt et...

...biologists and computer scientists learning and communicating with one another.      Currently our main focus...our worms.  We are on the lookout for genome modifications that do not exist in the CGC library but that...makeup of the bacterial species in the gut of the domesticated housecat, and seeing how that makeup differs...cats with the goal of optimizing the health of domesticated cats and learning about feline microbiomes. ...and the reagents are too expensive to make it practical - but we have members looking at how to re-use...

...Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering. CRISPR/Cas9 technology... addition to its utility in making targeted modifications to DNA, Cas9 can be reprogrammed to serve as... a gene of interest, they have been shown to dramatically alter levels of expression. Cas9’s ability to...Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease." Nature biotechnology ...Options for CRISPR Genotyping Cas9 Activators: A Practical Guide Additional Resources on Addgene.org Check...

...The vast majority of bacteria are undomesticated which limits the tools scientists can use to study them...the genome and is propagated during normal DNA replication and cell division. Mobile-CRISPRi does not disrupt...single-gene knockdown experiments. Mobile-CRISPRi applications Mobile-CRISPRi expands CRISPRi to non-model ...Elizabeth A., and Pamela A. Silver. "Harnessing undomesticated life." Nature microbiology 4.2 (2019): 212....PMC6424567. Peters, Joseph E. "Tn7." Mobile DNA III. American Society of Microbiology, 2015. 647-667. PubMed...

...efforts make a precise edit or insertion via a chemical modifier. We used these categories to see what... and the longevity of tools seen in Figure 3, indicates that the usefulness of popular CRISPR tools is...other countries, from Japan to Australia, are significant depositors and requestors. CRISPR tools are developed... into Addgene’s CRISPR data? Read our recent publication, “The Expanding Dissemination and Distribution...support for, this and previous Addgene CRISPR publications.  References and resources References Pyhtila...

...genes for drug discovery or to understand the intricacies of molecular pathways. These screens have gotten...diseases, as you can target your library to a significant degree using different types of AAV. Expression...into the genome, CRISPR may increase this rate. Specifically, portions of the AAV genome can integrate at...multi-plasmid system, where you have one AAV vector dedicated to expressing Cas9, while the other houses the...integration into CRISPR-induced DNA breaks. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-...

...BLAST sequence databases from the dropdown menu. Typically, we use the default nucleotide database “Nucleotide...Organism box. This small piece of information can significantly reduce your wait time for blastn, blastp, and...” program for blastn so that you can extract practically every single reliable base pair from your results... the “Align two or more sequences” option to drastically reduce waiting time for search results. Optimizing...other labs speed up their plasmid and cloning verification steps and free up more time for using your plasmids...

... of these techniques have in common? They all typically require secondary antibodies, frequently of the...in bacteria and extracted with common protein purification methods. Read on to learn more about nanobodies..., Alexa Fluor 568, or Alexa Fluor 647 before application to the sample. Staining with this multi-color...PubMed PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug...antibody-containing hybridoma supernatant is only $40, significantly cheaper than commercially available antibodies...

...transforming a plasmid into the bacterium Agrobacterium tumeficans - a plant pathogenic species that stably integrates...Agrobacterium tDNA insertion likely precipitated domestication of sweet potatoes (Kyndt et al., 2015)! It sounds...selection markers (basta, hygromycin, kananamycin, tunicamycin) (Nakagawa et al., 2007). Alternatively, if you...plants: Lots of plants produce lots of seeds. I typically use 8-10 plants per construct I transform. More...National Academy of Sciences of the United States of America. National Academy of Sciences, 112(18), pp. 5844...

...gives Snapgene great exposure to the community of biological scientists who use Addgene everyday. To get this...hesitation to ‘approach strangers’ and learn how to communicate outside my scientific expertise. Organizing a...will lead! Read our blog post about fostering communication in remote collaborations! Stay in touch: Maintaining...connection is to make sure you keep open lines of communication with colleagues who move on to new organizations...Universities themselves can help you do this through practical tools like alumni networks. I find it useful to...

...the Society for Advancement of Chicanos/Hispanics and Native Americans in Science’s (SACNAS) annual conference... written up in mSphere. A post-meeting survey indicated that the program was largely a success, but also... even more valuable program to Microbe 2020 in Chicago.  Finding our place in networking and mentoring... career scientists, students, and those from historically underrepresented groups. Through such mentoring...

... serotype, are particularly challenging. Their efficacy in any given system can be affected by any number... the Addgene repository can save researchers significant time and removes a variable from the lengthy ...available in the repository. GCaMP, a calcium indicator (biosensor, by Addgene’s categorization) was first...trends in our own repository data, giving us significant insight into which vectors are likely to be of...Waxmonsky, N. C., Swanson, L. E., & Fan, M. (2016). Practical Considerations for Using Pooled Lentiviral CRISPR...

...a reporter or tag that can then be detected.  Typically, the reporter is an enzyme such as horseradish...include undesirable molecules that may bind non-specifically to assay components and increase background ...staining. Direct ELISAs also lack the signal amplification afforded by secondary antibodies and thus are...antibodies are used.  Competitive ELISAs Scientists typically use a competitive or inhibition ELISA when measuring...While the precise setup of the method can vary, typically users first coat the multiwell plate with a reference...

...post was originally written by Joel McDade and significantly updated in 2022 by Susanna Stroik.  The advent... than ever to make precise, targeted genome modifications. Cas9 has been modified to enable researchers...uses a “nicking” Cas9, in which one of Cas9’s two critical enzymatic residues has been converted to an alanine...instead.  Choosing a Cas9 for site-specific modifications Site-specific deletions and knockouts Some experiments...Perhaps you need to introduce a single nucleotide modification at a defined amino acid to mimic a patient mutation...

...Why pseudotype? Lentiviruses and retroviruses typically target immune cells with their wild-type envelopes...out this review article published in Reviews in Medical Virology (Li et a., 2018).   Figure 2:...The glycoprotein will be incorporated into the replicating rabies virus envelope, travel retrogradely, and... Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene...Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons. Neuron 53:639–647 . https:/...

...addition, plasmids are typically prepared using endotoxin-free plasmid purification kits.     Figure...minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties... a concentration step prior to being frozen. Specifically, lentiviral particles are collected from the... Addgene's Data Hub Digital Droplet for AAV Quantification Viral Vectors 101: An Introduction to AAV References...

...Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate natural cellular...researchers can now select the best tool for their application from among many options.  Conclusion With these...engineered human uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467...via engineered uracil-DNA glycosylase. Nature Communications, 15(1), 6397. https://doi.org/10.1038/s41467...

...many cell types and often results in biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous...combining these two strategies allows you to specifically incorporate homo- or heterozygous mutations....in the field at the time but had never been systematically analyzed: introducing silent CRISPR/Cas9 blocking...efficiently incorporated during HDR. We decided to systematically characterize this phenomenon and found a general...University. He currently works to understand pathological mechanisms of Alzheimer’s disease by developing...

...even when larger vector genomes are packaged, the physical size of the genomes is still ~4.7kb. But despite...overlapping strategy has a region of homology, typically ~400-1400 bp long between part A and B of the ...overlapping split vector had an improvement in muscle physiological performance.  2. Trans-splicing Overview: For...and may need to be optimized for each unique application (Trapani et al., 2015).  Expression of smaller..., was 81% of that from a single vector, but significantly greater than the expression from an overlapping...

...favorite twitter #agarart accounts, Petri dish Picasso (@PetriPicasso) came together and delivered very impressive...are some of our 2017 contest winners:   Chicagoland's Community Biology Lab (@ChiTownBio) and one ...Mackin Lab from University of Colorado -Anschutz Medical Campus (@CSD_CUAMC) caught a case of holiday cheer...

...simple in C. elegans and bacteria. But CRISPRing typical mouse models, including creating Cre-dependent ...on both sides and the T7 promoter on one end. Typically, Easi-CRISPR works best with insertions of 2 kb...models varies from 8.5-100% of pups born, with typical efficiencies of 30-60%. Quadros et al. attribute...2.5x more animals than i-GONAD and is a more technically difficult process. Overall, the main drawback... out our CRISPR Guide Find Plasmids for Cas9 Purification Find CRISPR Plasmids for Mammalian Expression...

...any molecular biologist Tips for protein purification Replica plating Anti-CRISPR Retroviral vectors Career...source of information on recent advances in the biological sciences and techniques that simplify and expedite... help hone your blog post for our audience of biological scientists from a variety of different fields...

...Investigator III at Novartis, a multinational pharmaceutical company based in Switzerland. After completing...completing her PhD in Chemistry and Chemical Engineering with Harry Gray at Caltech, Crystal moved on to a ...Chapter 7: How Crystal developed her science communication skills and how they've helped her at Novartis...scientific teams at the Novartis Institutes for BioMedical Research to develop novel biologics for the treatment...

...Addgene podcast, we sit down with Alex Chavez, a Clinical Fellow in Pathology at Masachusetts General Hospital...Church and Collins labs at the Wyss institute for Biologically Inspired Engineering. Alex is the first MD PhD...and explains why he loved the first 2 years of medical school. 7:05 - 10:55 : Alex's focus during graduate...postdoc 12:01 - 13:21 : The Wyss Institute for Biologically Inspired Engineering and why Alex decided to...

...acquires through Addgene to produce molecular biological tools like Taq polymerase, T7 polymerase, EcoRI...scientists to continue to do excellent work in the biological sciences. Future awardees Now that this first...concluded, Addgene and Seeding Labs will be opening applications for a second round of plasmid awards on June...excited to discover what new and creative ideas applicants will propose for plasmids from the Addgene community...