...for mutation validation, we found these assays frustrating to work with, time consuming, and minimally informative...performed to amplify the target using PCR. Instead of tracking the reaction progress as in real time PCR, each...edit. Since exploitation of NHEJ to generate translational reading frame-altering indels for “functional... an unedited site distal tothe predicted double strand break, and a NHEJ/ “drop-off” probe designed to...to bind directly at the predicted double strand break site (Figure 2A). The single molecule resolution... 291–293 (2014). PubMed PMID: 24509632. PubMed Central PMCID: PMC4063274. 2. Findlay, S. D., Vincent, ...PubMed PMID: 26914317. 4. Mock, U. et al. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates...

...cell!)—ATP. There are biosensors for intracellular, extracellular, and even the ADP:ATP ratio. Most of... or animal system. This ‘thing’ could be a neurotransmitter, such as norepinephrine, or an electrolyte...last component of the biosensor is the signal transducer which will quantitatively give a readout for ...bio-recognition event is (fluorescence, pH, etc.). The transducer will generate a numerical value which represents...activated GFP expression (receptor), then the transducer could be a microscope, plate reader, or any other...sensors developed for it. Calcium triggers muscle contraction, and calcium channels are activated for neuronal...—keep reading to find out how!   Neurotransmitters Many canonical fluorescent as well as FRET-based...

...cleaves ssDNA in trans. In sum, CasPEDIA documents the target substrate (DNA/RNA and strandedness) along with...Cas enzymes) cleaves both strands of its dsDNA target in cis and has no trans activity. AsCas12a, on the...Cas enzyme: Nuclease properties (CasID): Cis/trans activity, PAM requirement, gRNA, and multiplex ability...features: CasID CasPEDIA will display both the cis and trans activity of each entry. For example, SpyCas9a (one...with its action on the substrate (nick, bind, cleave, mixed). This information represents the ‘big picture...required? CasPEDIA provides information on crRNA and tracrRNA requirements, along with any other required non-CRISPR-associated...common applications of an enzyme for your non-traditional experiment. Perhaps you are a structure junky...

... single strand break; when two such breaks are introduced near each other, in opposite strands. This “...Cas9 can be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses...Aprataxin, Tdp2) that function to make ends better substrates for ligation. Although we do not describe these...introduced. Repair of Cas9-induced breaks by NHEJ As illustrated below, NHEJ-mediated repair of Cas9-generated... the deletion can be less heterogeneous when constrained by sequence identities in flanking sequence (... “double nickase” strategy vastly reduces breaks and mutations at off-target sites. Christina Mork contributed....org/10.1371/journal.pgen.1000683 Bétermier M, Bertrand P, Lopez BS (2014) Is Non-Homologous End-Joining...

...quantity of your POI, but simply whether its concentration changes in response to experimental parameters...errors like uneven pipetting or irregular membrane transfer. Figure 1: A loading control is a type ...type of positive control necessary to demonstrate a POI’s absence. It also helps confirm that lanes are loaded.... No pipetter is perfect, and proteins may not transfer precisely evenly across the entire blot. So how... Figure 3: In a perfect world, protein concentration in your sample would directly correlate to protein... in the loading control are due to loading and transfer errors — not to any factors from before the proteins...you’ll need to conduct a control experiment to demonstrate that expression of your reference protein does...

... look like (the 3rd sequencing trace). While it requires a little extra wet lab work, this is still a ...your edit of interest. For all of the screening strategies outlined below, the technique can be used as ...do. A popular screening method for knockouts is Tracking of Indels by Decomposition (TIDE) (Brinkman et...et al., NAR). This technique uses the trace file generated in Sanger sequencing and a simple algorithm...Fig. 1: Overview of TIDE. The input is sequencing trace files of WT and edited cell lines. Then decomposition...and your Cas9-targeted cells. Simply upload both trace files to the TIDE tool online along with your sgRNA...design phase of your experiment for a screening strategy. Restriction enzyme screening is sensitive to ...

...MODAL) strategy." Nucleic acids research 42.1 (2014): e7-e7. PubMed PMID: 24153110. PubMed Central PMCID... reason. They offer many advantages over the traditional restriction enzyme cloning we once relied exclusively...generating long overhangs which allows the single stranded regions with homology to anneal a polymerase, ...hour or so, the sample is immediately ready to transform into competent cells. The master mix of enzymes...regions of homology. This video gives a helpful demonstration of how to use Snapgene’s program to design primers...Gibson assembly master mix for 1 hour, and then transform into competent cells. The success rate of this...if the ends of the fragments have stable single stranded DNA secondary structure, such as a hairpin or ...

...Signal transduction pathways are a lot like cell phone networks. Protein kinases deliver messages to ..., and how long their signals last all impact transduction of the message. To study signaling pathways,... game of telephone, often the signal must be transmitted by intermediate messengers. Optical control of...prone to aggregation in cells compared to the tetrameric Dronpa it was derived from. What are photoswitchable...photoswitchable kinases.  Below are a few examples demonstrated in Zhou et al. 1. Testing kinase inhibitors ...signaling by using psMEK1 and an ERK kinase translocation reporter with a mRuby2 fluorescent reporter ...feedback loops are a common feature of signal transduction pathways, but are often hard to decipher because...

...regulating its translatability." Science 343.6169 (2014): 419-422. PubMed PMID: 24458642. PubMed Central PMCID:... has recently published techniques to follow translation in living single cells. However, before CRISPR-Cas9...Both antibodies have a short antibody specific DNA strand added to them. Only if the proteins of interest...are in close proximity to each other can the DNA strands take part in rolling circle DNA amplification. ...When Eugenia is not by the microscope she likes to travel, discover new restaurants, go to concerts and bake...(2010): 897-905. PubMed PMID: 20543841. PubMed Central PMCID: PMC2920597. 2. Nathans, Daniel. "Puromycin...(1964): 585-592. PubMed PMID: 14166766. PubMed Central PMCID: PMC300121. 3. Söderberg, Ola, et al. "Direct...

...expression of the transgene of interest. In our studies, mCherry interfered with the transport to the membrane...versatile method to identify the expression of transgenes of interest. Furthermore, fluorophore variants...between them, could affect the properties or trafficking of the fusion protein (Kimple et al., 2013). ...different tag fused to the hM4Di might improve the transport of this DREADD to the plasma membrane. The haemagglutinin... important influence on the expression of the transgene of interest and its localization in the membrane... (Kimple et al., 2013) could help improve the transport of protein of interest the membrane.   ...Figure 1: Electron microscope images showing ultrastructural localization of hM4Di in monkeys. The pre-embedding...

... efficacious to target the coding or non-coding strand of DNA. As a rule of thumb, we avoid target sites...for HDR, the choice of target site is far more constrained by the desired location of edit; efficiency decreases...HDR for introducing edits. The same locational constraints are even more exquisite for the so-called base..., 2020). is not limited to single nucleotide transitions but still requires a nearby PAM, although these... modulating gene expression at the level of transcription – CRISPRa (activation) and CRISPRi (inhibition...most-efficacious to target a ~100nt window upstream of the transcription start site (TSS), while for CRISPRi, a ~100nt...of producing the guide (synthetic, in vitro transcription, or lentiviral delivery) can affect the accuracy...

...dinucleotide and methyltetrahydrofolate, which are universally abundant. Second, induction of transcription occurs...of customizable DNA-binding domains based on transcription-activator-like effectors (TALEs) as anchors ... thaliana. The results are light-inducible transcriptional effectors (LITEs) designed to bind specific...target them to specific cell populations. They demonstrated use of the LITEs to control gene expression .... Genes under LITE control showed a rise in transcription in as little as 30 minutes after light stimulation...light dose and is fully reversible after light retraction. Finally, because light can be applied non-invasively...really flexible and modular,” Konermann said. “Transcriptional activators are easy to expand or combine with...

...in an organism, including the embryonic and extraembryonic cell types. In mammalian development, cells...during ESC isolation. Low Chances of post-transplantation rejection when used therapeutically High Low...similar functional utility, iPSCs bypass the transplantation compatibility issues and ethical concerns of... used therapeutically, the chances of post-transplantation rejection are low. Further, as iPSC generation...somatic cell type to another, also known as transdifferentiation.   Many thanks to our guest blogger, Kusumika...and publishing after completing her postdoctoral training from Massachusetts General Hospital and Harvard...Beckmann, and W. Wurfel, Non-embryo-destructive Extraction of Pluripotent Embryonic Stem Cells: Implications...

...efficiencies, we could train our prediction model PRIDICT (Mathis & Allam et al., 2023). We trained our models in..., and deletions) without introducing DNA double-strand breaks (Anzalone et al., 2019; Chen & Liu, 2023...typically a Cas9 H840A nickase fused to a reverse transcriptase) together with a prime editing guide RNA (pegRNA...containing a primer binding site and a reverse transcription template (RTT). The design of the pegRNA has...learning-wizard colleague Ahmed Allam. Continued training led to the recent development of PRIDICT2.0 (Mathis...al., 2024). The additional datasets used in the training of PRIDICT2.0 make this model more suited for ...nicking gRNAs (used for PE3 and PE5 prime editing strategies) based on a DeepSpCas9 score ranking (Kim et ...

...Screening Kit Learn How the Clon Tracer Library Can Be Used in Cancer Lineage Tracing Resources on Addgene.org...each year. Despite these grim statistics, new strategies for attacking cancer are yielding promising results.... What is cancer, really? While cancer is extraordinarily complex at the molecular level, at the cellular...threat, cancerous cells still need to successfully infiltrate the circulatory or lymph systems to spread, or...model When mutations do arise and cancer ensues, traditional chemotherapies have taken advantage of the universally-dividing...University in Nashville, Tennessee has already demonstrated that a number of novel small molecules can bind...lines. Further, for each plasmid in the kit, the transcript most commonly found across all TCGA tumor samples...

...provided with a detailed training schedule that helped me transition easily. Training and responsibilities... was not as scary as it seemed. The transition into a traditional work environment was aided by the efforts...come without worry, as I had never worked the traditional “9-5” job. Prior to this, the only work experience...responsibilities Through this training, I was gradually introduced to Addgene as a company and given more details on...on what my position entailed. The official training process was lined out to span the first three months... what I was to do for my time at Addgene. This training prepared me for the independent work on the QC...concern to the plasmid function. The steps of the training process gave me enough time to become familiar...

...more “tractable” problems.  We must better define the postdoc (and even the grad student) trainee experience...such as new training programs (like at the Keck Graduate Institute), funding for training (such as the...speaking out and writing about issues in our science training infrastructure for some years.  He spoke eloquently... to choose institutions with allies engaged in training excellence to begin effecting change.  Panelists...Medical School, titled her presentation “How to Train Your PI”.   She focused her talk on the issues involved...advisors only know about academia and alternate training needs to be provided to take advantage of all ...journals.  He pushed for drastic change in the way we train scientists to start addressing this problem.  Finally...

...number of challenges ranging from long periods of training to limited job opportunities in academia - the...the main reason most people enter postdoctoral training. Similarly, there are several factors to consider...about how to get the most out of your postdoctoral training experience. If you're going to commit to doing...mentor is not always available for mentees due to travel, teaching, grant writing, etc. Also, it is unlikely...mentors. I found this beneficial for my projects, my training, and when I went on the job market. Additional... or that a reagent has not been convincingly demonstrated to work for your specific assay. "Quick and ...to replicate. Plan experiments logically and strategically to increase your rate of productive results ...

...disproportionately large number of honorees to have trained under a previous laureate (Zuckerman, 1977; Ma ...more than just technical knowledge, shaping their trainees’ very way of thinking too. Albert Einstein might... . . is not the learning of many facts but the training of the mind to think something that cannot be ...). But is it possible to bring the kinds of transformative educational experiences afforded to those fortunate...apprentice with top scientists to larger groups of trainees at an earlier point in their careers? A CURE ... to identify regions of MDH that interact with citrate synthase and presented their findings at the American... actively involved in the learning process. As trainees become more comfortable with the open-ended nature...

...use of antibiotic resistance DNA plasmids are traditionally grown in bacteria as a way to produce more of...in a plasmid. During the propagation and/or transformation process, some bacteria will either never receive... is nifty because the recovery period after transformation (the dreaded 60 minutes shaking at 37 degrees... available, cost-effective, timesaving for transformations. Cons: Prone to satellite colony formation ..., interchangeable with Amp, timesaving for transformations. Con: Expensive.   Kanamycin Kanamycin, or...selection gene. However, it does require a slower transformation recovery period than amp or carb (60 minutes...confers mammalian resistance to G418. Con: Slow transformation   Spectinomycin/streptomycin Spectinomycin...