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...). Advancing from this invention to KJ’s clinical application in under a decade is an astounding feat ...double-stranded breaks in the DNA backbone, making them significantly less prone to indel mutations that plagued CRISPR... safety of KJ’s experimental treatment, it was critical to reduce off-target editing as much as possible... ABE8e) would be tested for their base-editing efficacy in cellular models of KJ’s CPS1 mutation. Every...of identifying a successful editor. For this application, ABE8e turned out to be most effective. Targeting...editors with increased activity and therapeutic application. Nature Biotechnology, 38(7), 892–900. https:...

...clones, it’s wise to quickly assess whether a significant number of cells have been edited. For many experiments...enzyme screening for resistivity to digestion, indicating targeted edits.   Interested in this method...sequencing confirmation. All knock-ins A method applicable to knock-ins both large and small is a software...consider using a version of Cas9 that has been specifically engineered for reduced off target editing, such...Tao Chen, Jos Jonkers, Bas van Steensel. Easy quantification of template-directed CRISPR-Cas9 editing. (...and variant-aware in silico off-target site identification for CRISPR genome editing. (2019) Bioinformatics...Resources at Addgene: Read our CRISPRGuide Get practical help from our CRISPR Protocols Find gRNA Design...

...which to test the validity of your biological model and the technical performance of your assay. But, in... a more-typical scale of tissue culture while still offering the potential for true biological insight...components, the model system and the assay are just as critical for success. Sometimes, you might have a few positive...facilitating more rapid follow-up. We hope the biological community will find these reagents to be useful...

...al. "Systematic identification of signaling pathways with potential to confer anticancer drug resistance... therapies,” said Kris Wood of Duke University Medical Center. “While we do not yet have a comprehensive... of 17 signaling pathways that are frequently implicated in cancer cell proliferation, survival, differentiation... make the library usable for pooled screens. Applications of the cancer pathways ORFs kit Wood and his... he said. “It could [be used to screen for] morphological features, migration, or survival in different...

...Thousands of mutations have been implicated in cancer. Yet, the majority of them remain uncharacterized...annotate genetic perturbations that may play significant roles in cancer pathogenesis. Their work culminated... of mutations they identified included gene amplifications and fusions, missense mutations, and indels...mutations were found to be “neutral,” having no significant effect on cell viability compared to a wild-type...Approximately ~22% of the mutations, however, significantly increased cell viability, and these “activators...aberration drivers of pancreatic cancer." Nature communications 7 (2016): 10500. PubMed PMID: 26806015. PubMed...

...Since the first research applications of GFP were published in the 1990s, biologists have spent a lot... mammalian cells, and it may find wide use in biological research. Photoconvertible fluorescent proteins...color was observed after excitation at 488 nm, indicating that spontaneous conversion did not occur. Tobacco... Sattarzadeh et al., Sci Rep. 2015 CC BY 4.0 Applications and limitations of GFP photoconversion One concern...2):131-3.  PubMed. Find plasmids from this publication at Addgene. Subach OM, Patterson GH, Ting LM...nmeth.1664. PubMed. Find plasmids from this publication at Addgene.   Addgene’s Fluorescent Protein...

...scientific community support open sharing. But, one critical part was missing when the COVID-19 plasmids first...Carolina at Chapel Hill University of Illinois Chicago                     Boston Children's Hospital                           ...                           National Institute for Physiological Sciences Broad Institute                                       ...                                       Chinese Academy of Medical Sciences University of Texas at Austin University of California...part of research projects, another way is to systematically offer thousands of constructs for tagged and...enables research on solute carriers (SLCs) for medical research Recently, the Michael Davidson Fluorescent...

... QC/QA team as a DNA Sequencing Intern. The application process When I first saw the comprehensive job...immediately clear that Addgene wanted to ensure applicants understood what the expectations were for the...the intended position. After submitting my application, I was contacted to participate in a brief fifteen...the depositing labs intended. With the use of applications like SnapGene and Geneious, I was able to determine...team and shows how they fit into the system. Specifically as a co-op, it is encouraged to explore different...currently working on projects for the Science Communication function at Addgene. Working a 9-5 As it turned...

...natural and engineered repressible promoters. Chemically repressible promoters Tet-Off In its native context... GAL80 under different promoters and create sophisticated patterns of UAS-driven gene expression. To further... PMCID: PMC2942869. Find plasmids from this publication at Addgene. del Valle Rodriguez, Alberto, Dominic... PMCID: PMC2883883. Find plasmids from this publication at Addgene. Wei, Xing, Christopher J. Potter... PMCID: PMC3846601. Find plasmids from this publication at Addgene. Riabinina, et al. “Improved and ... PMCID: PMC4344399. Find plasmids from this publication at Addgene. Riabinina O, Potter CJ. The Q-System...expression of genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res...

...generally linked to dopamine (DA), a well-known reward chemical and neurotransmitter. But aside from DA’s famous...at a certain frequency, and this frequency can indicate behavioral state, for example, if someone is awake... it can be performed in a live awake animal by surgical implantation of the probe into a specific brain...in freely moving mice Record DA dynamics of optogenetically activated and inhibited neurons in live mice...neuronal imaging of dopamine dynamics with designed genetically encoded sensors." Science 360.6396 (2018): eaat4422... PMCID: PMC6287765. Sun, Fangmiao, et al. "A genetically encoded fluorescent sensor enables rapid and ...

...swimming where they periodically beat their tails left and right in a symmetrical motion to move forward...behavioral studies. In neuroscience, opsins are typically ion channels that allow charged particles to cross...Kolmer-Agduhr cells was sufficient to trigger the symmetrical tail movement unique to spontaneous slow forward... photostimulation in order to drive the full symmetrical response.  The function of Kolmer-Agduhr cells...Aristides B., Filippo Del Bene, and Herwig Baier. "Optical control of zebrafish behavior with halorhodopsin...

...technology, the Vale and Weissman labs took this biological lesson and created a system to amplify fluorescent...modified the GCN4 sequence to increase its alpha-helical structure and stability, creating v4 SunTag. Since...aggregation, it’s recommended for most imaging applications. What can you do with SunTag? Tanenbaum et al...flames,” it’s clear that the system is broadly applicable across many areas of research. Beyond fluorescence...activation of target genes, but we’ll save that application for another day! References 1. Tanenbaum, Marvin...et al. “A protein-tagging system for signal amplification in gene expression and fluorescence imaging....

... (e.g. 6xHis containing pDEST-HisMBP). After purification the protein can be released from the resin and...types of yeast expression vectors. Consider your applications and experimental design. You can create a new...more traditional approach of an autonomously replicating vector that carries either a wild-type version...expression pattern of a defined promoter region, ectopically expressing YGOI in a pattern that allows the ...a variety of other types of vectors for most applications used to study gene function. From making transgenic...transgenic animals, gene targeting and genome modifications to reporter vectors (e.g. pBV-Luc) and gene ...

... Foundation Round Table. In any field, good communication skills help when it comes to finding a job. ...an argument for taking the time to hone your communication skills. So, where can a graduate student or ...opportunities than ever before for scientists to communicate about their work and their interest in science...your area or at your institution; the news or communications office is a good place to start. It’s not unusual...about science writing as a career, the Science Communication Program at the University of California, Santa... your research stories. Consider practicing communicating your research by guest blogging here, just leave...

...problems, develop general technical proficiency, communicate your work, maintain ethical standards and interact...? Listen to our podcast interview with Harvard Medical School researcher Connie Cepko to learn about her... you admire her management style? Does he have ethical behavior? Does the lab encourage diversity? Knowing...websites listing alumi and it is easy to use publication records, university websites, ResearchGate and...

... at the same time. Barcoding libraries Just as physical barcodes help librarians track individual books...them useful for lineage tracing, among other applications. Cells can be identified using fluorescence,...sgRNAs to guide Cas9 to targeted genes. In these applications, however, nuclease-dead Cas9 is fused to a transcriptional...several coronavirus libraries have been used to systematically explore variations in the spike protein, teaching...: Preparing Pooled Libraries Pooled Library Amplifications Resources on Addgene.org Browse all pooled... to Plasmid Pooled Libraries CRISPR Library Amplification Protocol ...

... “species on species”) immunochemistry (IHC) applications an issue? The culprits are endogenous IgGs in...IgGs and your primary antibody, resulting in significant background staining (Figure 1A). Not all tissues...Figure 2B) Some labs have found that you can significantly decrease background staining by mixing your ...Randolph-Habecker J (2013). A Flexible Mouse-On-Mouse Immunohistochemical Staining Technique Adaptable to Biotin-Free...Partridge TA (1998). A New Blocking Method for Application of Murine Monoclonal Antibody to Mouse Tissue...Tuson JR, Pascoe EW, Jacob DA (1990) A novel immunohistochemical technique for demonstration of specific binding...

...the gRNA design process and rules for various applications is necessary going forward. The standardization...benefits to researchers conducting basic or early pre-clinical research. However, standard terms and techniques... I think I may have finally grasped just how complicated DNA repair pathways are. I fully admit to being...to me. But coming to #KSgenome and seeing the intricacies of DNA repair has emphasized how essential these...Alt-NHEJ), has been harnessed for CRISPR knock-in applications. CRISPR 101: Targeting RNA with Cas13a (C2c2...for CRISPR Genome Editing Other resources and publications CRISPOR gRNA design tool Researchers at the ...

...infection, because this setup allows for experimental replicates for each of the gRNAs. The idea was to assign...genetic tool for labeling just a fraction of a genetically-defined subset of cells. By labeling fewer cells...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ...measurements, or monitoring in vivo. To overcome these technical issues, Sabrina Büttner’s lab designed a calcineurin... reporter (blue) and stained with PI (pink) to indicate cell death. Image from Diessl et al., 2020. ...protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry. In comparison...

... (AmpR) and origins of replication (ori and f1 ori) which allow it to replicate.   How can I prevent...the bacterial selection marker and origin of replication. During bacterial culture, only the plasmid with...the antibiotic resistance gene and origin will replicate, and the transgene will be lost. Over time, bacteria...the recombined plasmid is still visible. This indicates that the DNA prep contains a mixture of the full...MK, Rudolph C (2019) Segmented poly(A) tails significantly reduce recombination of plasmid DNA without ...