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...genomic deletions. What are the advantages of Cas9 RNPs? There are many advantages to using RNPs for CRISPR ... Delivering Cas9-gRNA ribonucleoproteins  An advantage of performing CRISPR experiments using RNPs is... can be used to deliver a Cas9-gRNA RNP. This advantage has allowed scientists to perform in vitro and...et al. 2018). This was accomplished using a Cas9 mutant (Cas9M1C/C80S) that harbors two surface exposed...integration in crops, delivery of Cas9-gRNA RNPs to plants, as an alternative to plasmid based techniques,... account with these types of experiments. One important consideration is that the Cas9-gRNA RNP will be...it is likely that the use of RNPs will play an important role in its continued advancement. Andrew Hempstead...

...in close proximity and present in large enough quantities (and the correct stoichiometric ratios) to generate...generate useable data. What’s more, measuring and quantifying FRET is often easier said than done. Fortunately...overexpression of fusion proteins it requires can lead to aberrant subcellular localization of the studied PIPs. ...protein construction, cellular expression, and quantification (Kerppola, 2006, 2013). These are described... supraphysiological expression levels. BiFC quantification This is where BiFC outshines FRET — BiFC experiments...BiFC experiments. The ideal control is a mutated variant of one or both of your PIPs in which the protein-interaction...requires that you empirically test a number of mutants to identify those that inhibit BiFC in your cellular...

... as well as affinity purification with an anti-Cas9 antibody. These different tag systems and affinity...Using similar affinity purification with an anti-Cas9 antibody, the Li lab found a microRNA (miR483) that...transcription factor IRF-1. Immunoprecipitation, using an antibody targeting the 3xFLAG tag, allowed these researchers...this form, the CRISPR complex, consisting of a recombinant dCas9 protein and synthetic gRNA, is incubated...this technique, researchers have begun to make important discoveries in various biological fields. In 2015...chromatin by in vitro enCh IP technology using recombinant CRISPR ribonucleoproteins." Genes to Cells 21.4...

...from a sample. Chemical fluorophores can be substantially brighter and more photostable than fluorescent...reverting” to small molecule dyes seems daunting—no one wants to spend their days microinjecting fluorescent conjugates...systems consist of a genetically encoded enzyme variant tag that reacts specifically and irreversibly with... lipoic acid ligase, biotin ligase, phosphopantetheinyltransferase) – These enzymes catalyze the covalent...Fluorogen activating proteins (FAPs) – These modified antibody fragments bind and enhance small molecule fluorogens...excess dye from the sample, which is particularly important for samples where washing is difficult, such as...Nevertheless, these fluorophores were designed as antibody and oligonucleotide labels for fixed-cell imaging...

...between the purine bases (adenine, guanine, and hypoxanthine) or between the pyrimidine bases (cytosine, ... purine to a pyrimidine or vice-versa. Note: hypoxanthine (shown) is the nucleobase component of the nucleoside...have scientists solved this problem? By taking advantage of repair pathways within the cell — in some cases...Created with BioRender.com. Base editors take advantage of this single-stranded DNA (ssDNA) region, using...they expanded CGBE target scope by creating BE variants with other Cas9s that recognize different PAMs...processes that would cause DSBs, and its inclusion significantly reduced indels generated by CGBEs. What's next... aspiring genetic editors can now compare the advantages of base editing and prime editing, an alternative...

...in technical support including tips, advantages, and disadvantages of this overall rewarding role. Learn...high-impact journals, and the requirement for a high grant success rate. At this point, candidates are highly...the progress of the project. After that rather unpleasant experience I thought: I could do this job so ...additional data regarding the specificity of this antibody?” You will also help troubleshoot products, give...involved in such a role. In addition, it can be advantageous to speak multiple languages - that makes communication...possibly this same reason, it can be one of the more constant and safe corners in a company, less exposed to... in a Western Blot protocol and then blame the antibody when no signals are obtained. Or someone may heat...

...reporter can be measured and quantified to determine the presence and quantity of DNA, RNA or a mutation ..., scientists have utilized CRISPR-Cas9 protein variants, Cas13, and Cas12a, to develop simple, portable...fluorescent RNA by the “activated” Cas13 produces a quantifiable signal that indicates the presence of your targeted... pre-amplification step allowing for greater quantitation without compromising sensitivity. To detect... channel as the Cas13 reporter), they could significantly amplify the detection signal for a single target...CRISPR Trans Reporter Cas12a (Cpf1) is a CRISPR Cas variant that can also cleave dsDNA similarly to Cas9 (Zetsche...degraded at a rate of ~1,250 cuts per second. A quantifiable fluorescent signal designates the presence of...

... everyone who was doing the kind of work my lab wanted to do already seemed to know each other and what...and new friends while attending ASM Microbe in Atlanta, GA, in 2018. A lot of my experiences rang true...similar situations to themselves, allowed the participants to feel more comfortable at a large meeting....They really liked us.  The testimonials from participants in Binning Singletons at ASM Microbe in 2019...people to do in the pilot. We also found that all participants would benefit from more structure with regard...assistance than others, but we did have some participants feel like their mentors weren’t as invested ...expectations have been established for future participants regarding the activities and time mentors spend...

...not function properly in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF...make other edits. Once you have all the edits you want, cure pCas9 by growing the cells at 37 °C.     ... This would result in the lack of recovered transformants. Our speculation motivated us to update the ...the newly generated pEcCas/pEcgRNA system.   Advantages of the pEcCas/pEcgRNA system Compared with the...conclusion, the MUCICAT system has the following advantages: Fast and site-specific multicopy chromosomal...Sciences (CAS) Center for Excellence in Molecular Plant Sciences in 2019. She joined Sichuan Normal University...Lab of the CAS Center for Excellence in Molecular Plant Sciences since December 2020. His research focuses...

...however, in some cases it would be possible to grant those wanting to understand and/or attempt to reproduce...only ever a small fraction of all data directly relevant to a particular experiment, series of experiments...tool like an ELN. It is nevertheless critically important to be able to conveniently reference specific ...should have integrations with a broad range of relevant generic and science-specific tools. In addition...with a particular experiment or project thus significantly enhances the ability of others to subsequently...resources remain live. This means that someone who wants to query and attempt to reproduce an experiment ...Massachusetts, and feels privileged to be part of the vibrant life sciences research and commercialization community...

...bNLS and NES sequences. Disadvantages of KTRs KTRs also have some disadvantages when compared to FRET reporters...least one site. Phosphorylation is particularly important for regulating signal transduction and measuring...proxy measurement of kinase activity. The key advantage of KTRs is that they are easy to create and simple...fluorescence serves as a proxy for kinase activity.  Advantages of KTRs When compared to FRET reporters for measuring... measuring kinase activity, KTRs have two key advantages. First, they are easy to multiplex. Their design...together. Second, KTRs are easy to design. The key determinants of KTR function are the positions of the phosphorylatable...

...Nature Communications Find the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase fusion...fusion Caixia Gao’s lab has developed a new plant base editor from an evolved tRNA adenosine deaminase ... be used for A•T to G•C edits in protoplasts or plants of rice and wheat. Read the paper in Genome Biology...bacteria from various environments. It has the advantage of stable genomic integration and is easily transferred...three different Cas12a inhibitors. Some of these anti-Cas12a proteins have broad inhibitory effects on...human cells. Read the paper in Science Find the anti-CRISPR expression plasmids CRISPR-based method...recombinases used to excise markers. It has the advantage that CRISPR efficiency can be controlled by manipulating...

...tell only part of the story. This year, we made significant strides in making science more accessible and...serotypes, or hundreds of Data Hub reports detailing antibody experimental results. We also redesigned our website...growing collection of thoroughly characterized antibodies, and our contributions to key research papers...papers are already inspiring higher standards in antibody use (Ayoubi et al., 2024; Kahn et al., 2024). Building...materials — we're accelerating the future of science. Want to learn more about our latest developments? Subscribe... Laflamme, C. (2024). A consensus platform for antibody characterization. Nature Protocols. https://doi.org..., A. R. M., Cable, H., & Longworth, S. (2024). Antibody characterization is critical to enhance reproducibility...

...proteins (FPs) are also the foundation of multiple important technologies, including FRET and optogenetics....This simple technique has been shown to work in plant, Drosophila and mammalian cells, and it may find...was made by accident. After exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et...imaging process worked well with the common GFP variants EGFP, mGFP4-T, and S65T GFP, and no red color ...can be rapidly and efficiently photoconverted in plant, Drosophila, and mammalian cells. Photoconverted...enthusiasts! Addgene has empty backbones containing GFP variants used by Sattarzadeh et al., as well as a wide ...Addgene’s Fluorescent Protein Plasmids and Resources Relevant Blog Posts Plasmids 101: Green Fluorescent Protein...

...alpha2-AR antagonist, nLight sensors are not. Instead, they are blocked by alpha1-AR antagonist. This is...Neuromodulators like dopamine and norepinephrine have important functions in the brain but have been difficult...is important because alpha2-AR can provide inhibitory feedback in response to norepinephrine release, ...the nLight indicators. Speed One of the major advantages of this next generation of norepinephrine sensors...GRABNE, with both nLightG and nLightR showing significantly increased selectivity for norepinephrine over.... In particular, they’re focused on generating variants with improved affinity and dynamic range of both...

...through the ‘front door,’ the homepage is still important. It needs to express value, clearly communicate... what a site offers to their users, and most importantly, allow those users the ability to quickly access... to improve search and browse functionality. We wanted users to be able to find plasmids and viral vector...Along with improving search and navigation, we also wanted to emphasize the values that make Addgene unique...and allowed our content to dictate the space. We wanted to comfortably tell our story, to get scientists...500th time on addgene.org. Additionally we also wanted to highlight our blog, daily updates, and offer...conferences, on our website, or in social media, we wanted to make sure our friend matched our core branding...

...integrase mutants in the early 1990s (Leavitt et al., 1996), and since then, numerous variants have been...of the cellular DNA… forever!  This can be an advantage for researchers looking to generate long-lasting...there’s no telling which cells may harbor these unwanted interruptions. Insertional mutagenesis is especially...Transient expression of transgenes/shRNAs If you want your cells to express a protein or nucleic acid ...isolating genomic DNA from transduced cells and quantifying the integrated viral DNA along with a reference...detecting vector particle components or performing quantitative PCR titration of vector DNA in samples harvested... Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to ...

... and antibody labelling. In these experiments, biotin is added to a primary or secondary antibody that...organisms for life. Biotin is only synthesized by plants, most bacteria and some fungi, thus mammals must...of carboxyl groups to organic acids to produce important cellular metabolites. Biotin acts as a carboxyl...that targets your protein of interest or primary antibody respectively. Avidin bound to a reporter is then... on this complex can bind to your biotinylated antibody. This creates a large meshwork of biotin-avidin...of some other common applications and links to relevant papers and webpages are listed below: EMSA (Electrophoretic...de, Rodriguez, P., Bonte, E., Krijgsveld, J., Katsantoni, E., Heck, A., … Strouboulis, J. “Efficient biotinylation...

...help save time by screening out irrelevant candidates, and, more importantly, it’ll help you define salary...contributed by Damien Wilpitz founder and lead consutant at Experiment Designs Consulting, Inc. “How much...will take to secure your first (R-level) research grant.  Set your timeline from there, then start identifying...mouse experiments, start defining keywords you’ll want to find in candidates’ resumes or CVs; “vivarium...BEFORE searching for a faculty position. This is important to ensure that you’re looking at institutes that... try to keep conversations focused on research relevant objectives and their tangible resources. For example...Damien Wilpitz is a laboratory research management consultant and coach in Boston, MA. He specializes in assisting...

...run as The Green Lantern’s friend, St Walker: The Blue Lantern. While The Green Lanterns derive their power...Run4Rare team members complete the Boston Marathon, he wanted to run and play with my kids like any other youngster...Activin A. A commercially developed therapeutic antibody that binds Activin A and interferes with inappropriate...power from the emotion of will, The Blue Lanterns derive their power from the emotion HOPE; a fitting ...of rare disease sufferers like AJ. As the Blue Lantern Oath says: "In fearful day, in raging night, With...

...You've heard it before – it's important for scientists to get out there and talk about their work. To... is cool and why it really matters! There are important societal reasons, self-interested reasons, and...collaborative roles in academia, and that means it’s important to know how to talk about science to everyone ...myself) with a PhD in biology. Depending on what you want to do, that’s not necessarily bad news, but it is... understood why he cared to study mice on some distant island. He got a little money for his trouble, ...from the Ivory Tower.”  9)   If you think you might want to get serious about science writing, look into ...Communication Program at the University of California, Santa Cruz, has been turning scientists into confident...

...aligns with your interests. We've outlined some important factors to consider and summarize helpful advice...featured in "9 Tips to Achieve Success in Academia". Important factors to consider when choosing a research project... project After joining a lab, the next important decision to make is “What project will I work on?” In...like? What will be the impact on your field? You want to choose a research question that people (especially...critically evaluated your plan? Have you read the relevant literature to understand where the field is today...how can you mitigate them? If you were to write a grant for this project, what would be your critical experiments...typically require a diverse set of skills. It is important to know who in the lab and/or what facilities ...

...of Different Methods for Generating iPSCs. One important note: in this table, reprogramming efficiencies... to tumor formation in iPSC-derived and iPSC-transplanted mice. Cre-deletable or Tet-inducible lentiviruses... be used to package reprogramming factors. An important milestone of reprogramming is the silencing of...cells for the presence of transgenes is still an important control. Additionally, Sendai virus doesn’t rely...express the reprogramming factors of interest. The advantages of this method are that it’s relatively simple...removed.  Replicating oriP/Epstein-Barr nuclear antigen-1 (EBNA1) based episomes: These plasmids carry ...substituting uridine with pseudouridine. The key advantage of using RNA to reprogram cells is that it’s simple...

...immobilized on a reduced glutathione matrix. The advantage of a fusion protein is the efficient purification...of an already immobilized target protein. The disadvantage is that the GST moiety, which forms dimers, ...binding kinetics of the target ligand. Another important consideration is whether the affinity of an experimental...removal of the fusion protein could provide a more relevant kinetic analysis, but the trick is to utilize ...receptor and subsequently evaluated interactions with mutant and wild-type erythropoietin molecules. The in ...We are clearly NEB “fanboys” as the bacteria significantly enhance protein folding (tested using real science... rabbit (EMD, AB3596) and mouse (NEB, E8032S) antibodies against MBP are commercially available. Factor...

...Attainable, Relevant, and Time-bound. It is a good idea to take larger goals (e.g. Figure out what I want to do... the helpful accountability that is one major advantage of working with a mentor.The responsibility of...mentoring relationship really productive, the most important step you can take is to set goals to work on together...to help a Mentee reach goals. One of the most important contributions made by a Mentor is to help the ...graduate student – you get the idea. There are many advantages to being a Mentor: Learn other perspectives ...reap rewards if you put in the effort. If you you want more great advice on mentoring, click below to download...

...ideal for you. Here, we will chat about who might want to explore this role, how to seek out such an arrangement...them to a new institute and mentor that they may want to consider for future work placements. The chance...identified a gap or need in your research and decided you want to pursue a visiting position. What now? First, ... for the new placement. Make sure you read any relevant literature for the techniques you will be performing...these expenses, or you may need to seek out travel grants available from third parties or your home institute...generate data that will contribute to a publication? A grant? A patent? It is never too early to discuss authorship...feelers, apply to programs, and apply for travel grants. Good luck and happy travels! Resources Resources...

...CRISPR/Cas9 could be used to edit several autosomal-dominant mutations that have been connected to the early...Chinese Academy of Sciences found that mice with a dominant mutation in a gene that causes cataracts could... Cas9 mRNA and a single-guide RNA targeting the mutant allele (7). An independent team led by Hans Clevers... chose to explore its potential in mice with a dominant cataract disorder caused by a single copy of a...of the cystic fibrosis report. “This is a big advantage over induced pluripotent stem cells, which need...and many others. “Those off-target effects are important,” she said. “We also really need to get a handle...optimization. In addition, the current work in developing anti-CRISPR proteins aid the limitation of these mutations...

... within tens of minutes (Cabantous et al., 2013). The Shu Lab took advantage of this and came up with ...signals are often difficult to detect, and thus quantify, due to small fluorescence changes of the donor...protease activity (To et al., 2016). However, the lab wanted to increase the fluorescence range to better study...on protease activation. Normally, β10-11 are antiparallel. To prevent binding, the β strands in FlipGFP...sequence, β10 and β11 can rearrange to their normal antiparallel position and fluoresce. FlipCherry: Red fluorescence...FlipGFP and FlipCherry at Addgene. References Cabantous, Stéphanie, et al. "A new protein-protein interaction...

...studied for many decades, and they were known to be resistant to detergents and degradation under a variety ...different pH values. Using a screen that takes advantage of the fact that coiled R-bodies sediment in certain...see figure to the right), we identified R-body mutants that extend at both lower and higher pH. We initially...initially used this screen to find R body mutants that couldn’t extend at a normal pH and later realized...Plasmids expressing expressing both wild type and mutant R bodies can be found at Addgene here. What can...F. R. Organization and expression of genetic determinants for synthesis and assembly of type 51 R bodies...trait: Absence of toxicity of R-body producing recombinant E. coli on paramecia. European Journal of Protistology...

...have gained or lost a specific gene survive (ex: antibiotic resistance marker). In a previous blog post, ... your plasmid backbone, as it often relies on antibiotic resistance. But what about identifying clones...𝛽-galactosidase activity. These cells, presumably the cell you want, cannot break down X-gal and are white. Cells that...to be strategic about your enzyme choices. You’ll want to pick enzymes that will give you different sized...to amplify anything. Like any experiment, you’ll want the proper positive and negative controls for your...provide all of the information you need, so you'll want to design a custom primer. Because Sanger sequencing...wouldn't be reached by sequencing from either terminus. Want to know more? Visit this blog post for tips for ...