...We are excited to announce the recipients of this year’s Michael Davidson and Roger Tsien Commemorative...researchers, in addition to postdocs and graduate students as we have done in the past. Below, read about...Filip Tomaska and Tim Hanson, are characterizing variants of iGluSnFR3 in vivo and have used it to detect...post-transfection, excited at 505 nm (right). Scalebar represents 100 µm. Image from Abhi Aggarwal.   Brandán...providing insight into new methods for treating patients in recovery from opioid use disorder,” says Hubbard...line engineered to be electrically excitable. The mutants are co-expressed with a photoconvertible fluorescent...photoconverted, sorted, and sequenced to identify the mutants for further enrichment. Tian notes that this platform...

...Nature gives us even more to talk about, as it represents another step towards CRISPR germline editing ... the field will move forward from this work. Experiments & major findings Let’s start with the paper itself...forward with gRNA 1 to conduct microinjection experiments in S phase zygotes at 18 hr post fertilization...are needed for proper development. Ma et al.’s experiments showed a 3% mosaic rate, and this rate would ...to correct the hype when we see it. This paper represents a huge advancement in our understanding of how...perform in embryos but also shows that many more experiments are needed before we can consider clinical applications...applications for germline editing. Although this paper represents a large step forward in the CRISPR field, it’...

...that stability of the RNA used in CRISPR-Cas9 experiments – the “gRNA” – suffers from the same issues as...nuclease-resistant, resulting in less off-target events. While both have similar advantages, bridged nucleic... Editing). This is particularly important in experiments in which viability is being compromised by introduction...CRISPR-Cas9 system can initiate targeted editing events. Both technologies utilize chemical modifications...gRNAs? Dye labeling the RNA in your targeting experiments allows you to visualize transfection efficiency...efficiency and even track localization in cellular experiments. There are many suitable dyes to choose from ...-703 (2020). https://pubs.acs.org/doi/10.1021/acscentsci.9b01093 More resources on addgene.org  View ...

... from their native regulatory elements. Controls for RaPID experiments RaPID negative controls include...interest. See figure 1 below for details on the key components of RaPID. Ramanathan et al. also tweaked BirA... the role played by RNA-protein interactions in events that occur on a short timescale, such as during... BirA*-based affinity purification mass spec experiments. These background proteins were then subtracted...correlated with poor survival in breast cancer patients. By digging into the literature and analyzing ...disease. RaPID can also be used to test how RNA variants associated with human disease affect protein binding... with increased serum iron levels seen in HHCS patients. These are just three examples of how RaPID can...

... about postdocs. Among other involvements, he helped to establish grants through the Sloan foundation ...biomedical workforce. Quotes taken directly from participants (summarized in 2) touched upon many problems...hired to produce results, not scientists”  These comments are well correlated with published data. For example...better solution would be to reduce the number of entrants into PhD programs to more closely reflect the ... market for PhDs Later this week, postdocs and students will again gather in Boston - this time with the...arguing against reducing the number of graduate students accepted in STEM fields (6). Melanie Sinche ...PMC4484856. 5. Bourne, Henry R. "A fair deal for PhD students and postdocs." Elife 2 (2013): e01139. Pubmed ...

...(electronic table of contents) alerts from journals you read often. The contents of each issue will be...explain some of the rationale behind the line of experiments, what data they collected and what it means. ... model or overall conclusion. In others, the experiments are geared towards eliminating possibilities ...deeper about the data they are showing. Are those experiments the best way of figuring out what’s going on?...controls? Refer back to the methods section for finer points of the protocols. Are there any red flags about... days of file cabinets filled to the brim with reprints and papers with scribbled notations in the margins...searching, reading and organization tips in the comments below. Additionally, read more of the Beginner...

...Many neuroscience experiments that require gene expression in a specific cell type rely on transgenic...of off-target expression and ways to optimize experiments to minimize it. Overview of DIO/FLEx vector design...problem of DIO/FLEx vectors, the cause of these events, until recently, was not known. A recent paper ... both the serotype and promoter with in vivo experiments to determine the optimal/minimal injection titer...interest. You should now be able to make smaller adjustments to a set of injections into your animal model...may be tempting to skip some steps and get to experiments. However, by optimizing all conditions first,...results.  Conclusion Using AAV vectors in your experiments requires a full understanding of vector performance...

...). If you are wondering which virus fits you experiments best have a look at this viral vector overview...and expression of these vectors when used in experiments. Retroviral vectors make up a large toolbox ...screening of thousands of genes in genome-wide experiments. Gamma-retroviral vs. lentiviral vector systems.... More detailed descriptions of the lentiviral elements and the 2nd and 3rd Generation Lentiviral Systems...and tips for using retroviral vectors in your experiments. Gamma-retroviral vectors Popular gamma-Retroviral...can be used to induce site-specific recombination events and is widely used in the field of mouse transgenics...Zhang lab: We are constantly improving our CRISPR reagents. Please check www.genome-engineering.org for the...

... viral components or the transgene itself are used to quantify the number of integration events. Another...common assays for physical titer are direct p24 measurements via ELISA and qPCR for viral RNA. Measuring ...quantified using qPCR primers targeting specific viral components such as LTRs, gag, WPRE, antibiotic resistance-genes...genome and a cell that has had multiple integration events. In addition, promoter choice can also impact transgene... approach can discriminate multiple integration events, reducing the possibility of underestimating the...titering as this will better represent future experiments. The aforementioned titering options all have...of infection (MOI) for predicting gene transfer events." Genetic vaccines and therapy 2.1 (2004): 1. PubMed...

...the suggested amount, instead of the other dry ingredients above. Media without growth (top) and with growth...

...generations. Figure 1: Mobile-CRISPRi's modular components. Image from Peters et al., 2019 with permission...using the bacterial integrative and conjugative elements in Bacillus subtilis (ICEBs1) system. ICEBs1 carries... Top: A Tn7 transposon containing the CRISPRi components and a plasmid with the Tn7 transposition genes...lacking DAP select against donor bacteria and for recipients with an integrated copy of CRISPRi. Bottom: An... An ICE element containing the CRISPRi components is transferred to recipient bacteria by bi-parental ...suited for genome-wide CRISPRi library screening experiments where maintaining library diversity is important...rates are only suited for single-gene knockdown experiments. Mobile-CRISPRi applications Mobile-CRISPRi expands...

...introduce their students to this otherwise invisible molecular world. For the students, the models force...hand-deliver the model, the following sequence of events plays itself out: The researcher breaks eye-contact...membrane of the endoplasmic reticulum. And graduate students, momentarily forgetting that the physical model...Subscribe to CRISPR Blog Posts How do educators and students use molecular models?   We created the Center ...instances of focused attention become magical teaching moments. The models function as thinking tools – eliciting... Team modeling program in which small teams of students work closely with local researchers to learn about... designed to make the molecular world real for students. Many thanks to our guest blogger Tim Herman!...

..., safety requirements and expression limitations may be too constraining for some experiments, particularly...that often affect which is chosen for certain experiments. AAV can be safely handled at BSL-1, while lentivirus...vectors is how the cargo — in this case, the CRISPR components — are expressed. Unlike lentivirus, cargo delivered...AAV genome can integrate at double-stranded break points caused by CRISPR-Cas9 editing. The good news is...the amount of cloning, another option is to use variants of Cas9, like split Cas9, or a smaller Cas9 ortholog...tissue-specific diseases has grown exponentially, aided by advancements in in vivo CRISPR screens. AAV vectors have ...have played a large role in these advancements, due to their many benefits for in vivo models. Over the past...

...contamination free environment for successful experiments: To clean air and beyond Turn on the hood for...wipes) before and after your work. Keep your contaminants away While it’s likely that you’re the main ...main source of contamination for your experiments, the bright side is that you also have the power to take...aerosols that may enter the hood. Planning your experiments thoroughly before entering the hood is another...microscope will help you identify bacterial and yeast contaminants. Set up a routine to split cells Don’t be lazy...rendering their continued use inadvisable for experiments requiring reproducible results. Most cell culture...should not be used for important transfection experiments right after they’ve been thawed. Additional passages...

... mutually exclusive elements. Labs have successfully utilized 2A and IRES elements within the same multicistronic...for research purposes are described below. IRES Elements Translation in eukaryotes usually begins at the...translation initiation independent of the 5’ cap.  IRES elements are very useful and commonly found in bicistronic...however, they do have some disadvantages. These elements are quite large (500-600 bp) and may take up precious...express more than two genes at a time using IRES elements. Further, scientists have reported lower expression...features and spend more time developing the clever experiments and innovative solutions necessary for advancing...ITAMs ensures effective negative selection and prevents autoimmunity. Holst et al (Nat Immunol. 2008 Jun...

...colors and 10 points to keep in mind when choosing an FP (or two) for your upcoming experiments. Fluorescence...FPs just keeps getting larger with hundreds of variants available. Read on to familiarize yourself with...emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP (CFP), and yellow FP (YFP...YFP) were derived. For a breakdown of GFP, it's variants, and their relevant mutations, check out Marcy's...Bimolecular Fluorescence Complementation (BiFC). 8 points to keep in mind when choosing a fluorescent protein...you are planning to express the FP in acidic environments (e.g. yeast cytosol, which is slightly acidic...with very comprehensive data sheets on many FP variants Plasmids 101: Green Fluorescent Protein (GFP) ...

...in high pH environments and is less susceptible to degradation in agar plates. This prevents satellite ...less stable at high temperatures and in acidic environments.   Carbenicillin   Carbenicillin, colloquially...genomes. If you need a plasmid that can go between plants and bacteria, it’s a great option! Otherwise, since...eukaryotic cell culture, yeast, bacteria, and even plants! The zeocin resistance gene is present in bacteria...et al). Thus, it may not be a good choice for experiments in which off-target mutations are undesirable..."Check icon" %} Zeocin Excellent No Bacteria, plants, and eukaryotes Not Tn5-containing cells {% icon...temperature and pH on the stability of antimicrobial agents in MIC trays. Journal of Clinical Microbiology,...

...sample is pure and free of any contaminants that could affect the measurements. You'll need to prepare the... or very similar buffers if not, since buffer components can affect the final readout. When preparing ...duplicate or triplicate. The closeness of the data points generated by replicate values provides useful information...diluted standard is plotted against its OD560 measurements and the trendline y = 0.6561x + 0.019 calculated...predicate values and measures how well the data points fit the trendline. R2 ranges from 0 to 1 with 1...approaches the minimum and maximum thresholds. If the measurements for the unknown sample fall outside of the dynamic... to set yourself up for success!  Study the instruments and specific assays you will be using ahead of...

...(genomic DNA and RNA), proteins and chemical contaminants that have been used during the purification ... indicators of DNA purity are the absorbance measurements at 230, 260, and  280 nm. The absorbance ratios...scientists an indication of purity versus protein contaminants. The 260 and 230 nm ratios should fall ~2 - ...provide an indication of purity versus chaotropic agents as guanidine thiocyanate and guanidine hydrochloride...extraction of enough plasmid to last for multiple experiments. Another fundamental aspect for transfection ... being free of any nucleic acid and chemical contaminants, these DNA samples must also be entirely endotoxin...ultimately affects the quality and outcomes of experiments. For these reasons commercially available kits...

...directed evolution approach with some notable improvements. Directed evolution techniques mimic natural...utility of the system for use with low affinity variants of TEV. The new system has been demonstrated to...the evolution platform to generate TEV protease variants. Catalysis by TEV protease allows a transcription...fluorescent tools for labeling structures and compartments in mammalian cells By Angela Abitua Genetically...are often used to label  specific structures, compartments, or specific localization of biomolecules within...plasmids at Addgene The MoClo CRISPR/Cas Toolkit for plants includes 95 plasmids consisting of CRISPR/Cas nucleases... allow for sensitive and high spatiotemporal measurements of norepinephrine. These norepinephrine sensors...