... SCF E3 ubiquitin ligase complex. In the presence of auxin, this TIR1 complex binds to the degron sequence...depiction of chromatin within a nucleus -> DNA compaction around histones -> Examples of histone-modifying...included for visualization purposes, but the key component of the editing assemblies are sensor-actuators...The tool-kit consists of highly useful vectors compatible with Gibson and Saptrap cloning systems to generate...Resources page.  Plasmids for delivering CRISPRi components by a single AAV vector are now available from... developed by Taekjip Ha’s lab where Cas9-gRNA complex binds the target DNA without cleavage until it'...

...-Vvd showed substantially improved activity as compared to other recombinase/photodimer pairs. Created.... https://pubmed.ncbi.nlm.nih.gov/24390935/ Hypercompact genome editor CRISPR-Casɸ By Melina Fan The ...toolbox with their discovery of CRISPR-Casɸ, a hypercompact genome editor from the Biggiephage clade of ...cells. Casɸ from Biggiephage can eliminate competing mobile genetic elements. Image by Basem Al-Shayeb...the Doudna Lab. Scientists looking for more compact genome editors due to delivery constraints and ...efficiency and reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants...in screens without any reduction in performance compared to larger genome-wide libraries. Find the library...

...increased versatility in probe attachment, and compatibility with far-red and near-infrared fluorophores....enable highly specific visualization of cellular components, typically a protein of interest. Fluorescent...ligands Effects on protein function and cellular components: It's important to assess how dye-conjugation... function and its propensity to bind cellular components. Some dyes target specific organelles like mitochondria...may prove challenging to stain intracellular compartments in in vivo model systems, so extra care must...Transfer (FRET) applications, the dyes need to have compatible excitation and emission profiles to enable effective...finding the right dye for your experiment more complicated. We hope this post helps you in your search for...

... a Cambrian-like explosion of software tools accompanying the CRISPR revolution. (See omictools for even... a 96-well plate and comes out of it. Biotech companies are known to have tools that do this internally...guides and primers in batch, optimized for HDR. Compared to wealth of resources for guide design, there...batch analysis because of its ease-of-use and comprehensive reports. With CrispyCrunch, you get the all ... ranking formula that does just this: Rank = (compressed CFD score) * (gaussian normalized cut-to-insert...is within the coding region, the algorithm then compares the guide sequence to every possible 23bp sequence...Francisco. Before starting his career in software, he completed a MSc in Psychology at McMaster University in ...

...derived from AAV9, their efficacy is generally compared to the AAV9 serotype or an engineered precursor...doesn’t lead to enhanced transgene expression, compared to AAV9, in BALB/cJ mice but is more effective..."Mouse Strain-Specific Tropism?" column. For a complete list of mice strains with LY6A-dependent tropism... by the Gradinaru lab (Deverman et al., 2016). Compared to AAV9, it effectively transduces neurons and...toward astrocytes, but its neuron transduction encompasses more molecularly-defined neuronal subtypes and...astrocytes and neurons, with greatly increased efficacy compared to AAV9 (Hanlon et al., 2019). This capsid was...brain regions, with reduced off-target expression compared to AAV9 in infant rhesus macaques. The AAV.PAL...

... stranded DNA breaks that commonly result in a complete loss of function while nuclease-deactivated Cas9...have several limitations. CRISPRko results in a complete loss of function meaning that if an essential ... more hits than the SAM library approach in a comparison study screening for vemurafenib resistance in...(Konermann et al., 2015). Root and Doench also compared the Calabrese library to screens performed with...that ORF and CRISPRa libraries can be used in complement to study gene function. Find the Calabrese pooled...gene function while using fewer sgRNAs. These new compact libraries will allow scientists to conduct genome-wide...transcriptional activation by an engineered CRISPR-Cas9 complex." Nature517.7536 (2015): 583. PubMed PMID: 25494202...

...Construction of solid matrix-antibody-antigen complexes containing simian immunodeficiency virus p27 using...structures of CRBNmidi alone (A) and in a ternary complex with the multiple myeloma drug mezigdomide and ...on its own, with drug ligands, and in ternary complexes with other proteins, establishing CRBNmidi as ...matured considerably faster, with expression levels comparable to GFP during early embryogenesis (Figure 3).... through the middle of each embryo are shown, comparing GFP, wrmScarlet (mScarlet), and the new mScarlet3...Merritt D.M., Pe K., Cesar M., Hobert O. (2024) Comparative analysis of new mScarlet-based red fluorescent...than FRET-based mechanosensors (~60 kDa) and are compatible with common microscopy methods. Plus, the luminescence...

...mutations in the genome, but they’re inefficient compared to CRISPR/Cas9 edits that rely on double strand...only occur through editing by a APOBEC-Cas9n-UGI complex. So how exactly does this work? Figure 1:...editing efficiency can be easily determined by comparing cells expressing only eGFP expression to cells...DNA editing efficiency of APOBEC-Cas9 editisome complexes in real-time (St. Martin et al., 2019). To create...DNA editing efficiency of APOBEC-Cas9 editisome complexes. The eGFP reporters rely on the correction of ...frequency of genomic base pair edits of interest compared to cells segregated solely by a reporter of transfection... single base editing by APOBEC-Cas9 editosome complexes. Scientific Reports 9: . https://doi.org/10.1038...

...neuronal function, with far fewer side effects compared to lesioning methods -  they may be the key to...camera or tracking software. Locomotion is a highly complex process on the level of individual neurons and ...different motivation (and brain areas associated) compared to an animal running to escape a threat. Locomotion...plus maze, the light-dark box is a simple device comprised of a brightly lit (risky) and a dark (protected... control group preferred staying in the dark compartment (Howerton at al. 2014). Addgene has several recombinase-expressing... passive mode, the stronger is the depressive component of the animal behavior. With the availability ...behavioral neuroscience to reveal insights into the complex relationship between genes, neurons, and behavior...

... newly formed compensation committee. The compensation committee creates a compensation philosophy that...employee compensation and also serves as an objective body to oversee some of Addgene’s compensation decisions... environment.   What do you hope Addgene will accomplish in the near future? Right now it’s a matter of...

...websites compiling antibody data that can help you compare antibodies from different companies, such as... a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence and indirect....  How do you find validated antibodies? Many companies that produce and distribute antibodies validate...through their publications or as a review on a company website. It is important to review the data and... a couple of antibodies that seem promising to compare in your own lab. Even with all of the resources...

...the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences, Cas12a activity ... amplified region of interest. Cas13 uses a complementary crRNA sequence to bind to the sample and upon...steps and the Cas protein used. Here, all the components needed for the reaction are mixed in a single...incubated at one temperature. Two Cas12a-crRNA complexes are designed, each binding to one strand of the... RPA primers, the Cas12a-crRNA binds to the complementary region in the target sequence and this activity... substrates for Cas13. Cas13, upon finding complementarity between the gRNA and a given fluorescently ...streptavidin coated beads. Fluorescently labelled dCas9 complexes containing sgRNA that have no mismatches bind ...

...are designed to be complementary, but each primer may extend beyond the complementary region as long as...products (green) are digested with enzyme “A” as compared to the products of parental plasmid digestion ... Primer Design As a rule of thumb, 11 bp of complementary sequence on either side of the desired mutation...template should be isolated from a methylation competent bacterial strain (e.g. DH5α which is dam+). Methylation...A-overhangs (a feature used in TA-cloning). Such non-complementary bases at the 3’-end interfere with plasmid reconstitution...that any residual parental plasmid (usually from incomplete DpnI digest) can also form colonies under these...

...5). Optimization of this system defined three components required for translation (Figure 1): A tRNA ...the pSer-incorporated polypeptide. These three components were shown to effectively incorporate phosphoserine... the phosphoserine OTS, Sep-OTS, contains the components required for the translation of pSer-incorporated...host strain for pSer-incorporation The second component necessary for phosphoserine incorporation is a...enzyme function, protein interaction, etc. by comparison to non-phosphorylated or ‘wild type’ control ...substitution. Alternatively, you can express the completely wild type protein from the same plasmid.  Upon...dephosphorylation reaction can be tracked through the comparison of phospho:non-phospho species on PhosTag gels...

...lengths to keep accurate records, but given the complexities of modern research, it’s often not enough. A... have software tools that help us efficiently accomplish our daily goals. These tools remind us of our...others using excel sheets or organizing folders in complex ways, there are solutions that bring more to the...-based solutions or open source solutions. In comparison to paper-based record keeping, ELNs offer functionalities... if you are working for a large institution or company, site licenses might be available as well. In most...information. Does your lab need to meet any specific compliance requirements – e.g. 21 CFR part 11, HIPAA? Answering...of time and effort and give the lab a serious competitive advantage.  Many thanks to our guest blogger...

...transform into competent cells. The master mix of enzymes can be purchased from a company (e.g. NEB or ...so the entire reaction takes an hour or less to complete at 50 °C. After an hour or so, the sample is immediately...master mix for 1 hour, and then transform into competent cells. The success rate of this reaction is typically...terminator sequence), since this would directly compete with the required single-stranded annealing and...ensures correct assembly order of the fragments as compared to the smaller overlapping sequences left with...assembly meets CRISPR Gibson can be adapted to more complicated cloning schemes, such as those where the vector...

...credit for what we have accomplished. Starting with a recap of accomplishments, from learning new techniques...tangible research and career goals, and celebrate accomplishments. These techniques are laid out in Vincent et...conversation. Goals and Planning Worksheet: Lists accomplishments, research/professional goals and feedback. ...including major project goals and effort needed to complete them. These documents dictate the subject matter...concrete plan in hand, trainees are motivated to complete their work rather than overwhelmed or confused...

...services don’t offer plastic recycling, specialized companies can recycle plastic-- you just have to find them...product packaging) for free! It has to be from their company and it has to be packaging (no coated tissue culture... label. Recycle nitrile gloves Terracycle is a company that takes items not commonly accepted at recycling...your lab or department and send them back to the companies for reuse. They’re really easy to spot and even...recycling program at your institute! Universities and companies have the potential to make a huge impact directly...! These days, it’s important for individuals, companies, and universities to be sustainability leaders...

...methylation competent E. coli for this procedure to work properly. Directly transform chemically competent cells...tips for applying it in your own lab. Table 1: Comparison Between Standard Restriction Cloning and Fast...are then transformed directly into chemically competent E. coli and transformants selected with the appropriate...insert with an additional 15-17 bases that are complementary to the overlapping vector ends. See an overview...an hour at 37°C. This should be enough time to completely digest the template DNA. Note—templates must ...

...1]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The piggyBac...also enables the excision of the transposon in a completely seamless manner, leaving no sequences or mutations...enhanced random integration efficiencies that are accomplished with transfections and are therefore virus-free...enhance gene editing? Gene editing can now be accomplished in a more cost-effective and efficient manner...challenges still remain, such as making gene edits completely seamless (i.e., so no other genetic alterations...transposase (PBx), which contains mutations making it competent for excision, but defective for integration [4...   How does the piggyBac™ gene editing system compare with other methods of gene editing? There are primarily...