...Named for the "stellar explosion SUperNova," SunTag can help you turn up the brightness in your fluorescent...protein is to fuse it to a fluorescent protein. You can then study where the protein is localized, and how...Having seen that mitochondrial tracker GFP-mitoNEET can impair mitochondrial function, they examined the ... recommended for most imaging applications. What can you do with SunTag? Tanenbaum et al. performed a ... of different experiments in their paper, and so can you! In almost any case you would use a traditional...kDa for GFP alone. Although traditional FP fusions can also affect protein activity, half-life, or localization...many areas of research. Beyond fluorescence, SunTag can also be used to improve CRISPR-based activation of...

...that is fused to a nicking Cas9. This base editor can be used for A•T to G•C edits in protoplasts or plants...regulated not only temporally with doxycycline, but it can also be regulated spatially with Cre-recombination...backbones, and gRNA structure, numbers, and promoters can all be chosen. Read the paper in PLoS One Find...Mobile-CRISPRi” plasmids, a set of modular CRISPRi tools that can be used in diverse bacteria from various environments...broad inhibitory effects on Cas12a orthologs and can block Cas12a-mediated genome editing in human cells...markers. It has the advantage that CRISPR efficiency can be controlled by manipulating complementarity between...

...insert, liquid media can be faster and easier to work with. In either case, it can be useful to run a restriction...downstream applications. As individual libraries can vary significantly, it’s important that we have an amplification...because repeated amplifications of pooled libraries can lead to issues such as recombination and loss of ... for smaller libraries, chemical transformations can also be used. Efficiency for chemical transformations...your lab’s electroporator uses, etc. The DNA amount can be anywhere from 10 ng to 1 μg of DNA. It often correlates...individual electroporations. The specifics of this can be based on the manufacturer’s protocol for transformation...(on plates). In general, amplification on plates can help to ensure that diversity of the library is maintained...

... inexpensive and can be 5’-phosphorylated in house using a simple PNK reaction or can be purchased 5’-...required for its optimization and development before it can truly be pitted against CRISPR head-to-head.  Exciting...rather than an RNA guide as with Cas9. Third, it can be used to target “difficult” GC-rich regions of ...protein (probably while it is being synthesized) can form a complex with an ssDNA guide. Therefore, researchers...versatility of its delivery methods. Researchers can conveniently transfect/inject cells with in vitro...achieved by a few respondents suggesting that NgAgo can indeed be optimized for genome editing in mammalian...time is required for NgAgo optimization before we can make any major conclusions: That said, many people...

... you can do this. If you are going to complete your colony PCR analysis in the same day, you can save ... on an LB plate. You can use this plate to start liquid cultures. Lastly, you can start small overnight...with restriction enzyme digest; however colony PCR can accomplish the same thing in less time and for less...clone without the insert. This type of primer pair can tell you if the insert is the correct size and whether...the size of the insert and if it's in the plasmid Can be used to screen clones that were created with the...Taq polymerase is sufficient. Controls: Controls can make or break an experiment. The best controls for... insert. These controls are quick references you can use when you run your PCR products out on a gel to...

...research can be a daunting challenge. With over 80,000 deposited plasmids from around the world, it can be ...Vector Type” you can filter by experimental purpose (e.g. CRISPR) Beyond search What can you do if you ... through to a different part of the website, you can now use the pulldown menu on the search page to quickly...screenshots show the range of different terms we can use to find materials from this publication. Searching...Searching for the title of a paper works You can also find the associated plasmids through the article’...primary investigator where the plasmid was created can narrow your search results to exactly what you need...Availability to Industry: Restrict search to plasmids which can be distributed to industry research scientists Viral...

...is constantly evolving. Variants of Cas proteins can be used for genome editing, activating gene expression... and well studied CRISPR system, N. meningitidis can also be used for genome editing. In this study, the...control samples to 0-10% in Acr samples. Acr proteins can also be used to prevent dCas9 tethering to DNA. Since...specific to a particular Cas protein while others can inhibit CRISPR enzymes from multiple species of bacteria...organized by the type of CRISPR-Cas system they block can be found here. How do anti-CRISPR proteins work?...). Figure 1: Anti-CRISPR proteins (Acr's) can block CRISPR activity in many ways such as inhibiting...Reduce off-target effects Prolonged Cas activity can increase the chance of off-target editing particularly...

...: Cut: CRISPR can be used to cut the DNA sequence. With a repair template, CRISPR can be used to introduce...100% specific and can cut DNA sequences that researchers don’t intend it to. CRISPR cannot cut all sequences...DNA. Fusing the nuclease to an adenosine deaminase can convert adenosine to inosine. Activate/Repress: Modified...proteins continue to expand the targeting range. CRISPR can be difficult to deliver. Not all cells efficiently... Modified versions of CRISPR nucleases that can’t cut DNA (dCas9, for example) that are used to increase...

...function downstream of DNA, such as RNA interference, can reversibly exert their effect independent of gene...of different small RNAs (Figure 1). These siRNAs can exert their gene silencing effect through targeted...sequences (Figure 1). As a result, gene silencing can be easily induced by exogenous application of long...machinery. Alternatively, endogenous miRNA precursors can be engineered to created artificial miRNAs. The processed...CRISPR-generated mutants believed to be knockouts can maintain partial function due to the use of exon ...trigger RNAi activation. A typical RNAi experiment can be divided into three fundamental steps: creation... (Figure 1). With this technique, multiple genes can be simultaneously silenced if a consensus sequence...

...your experimental RNA. To solve this problem, gRNAs can be chemically modified to resist degradation while... Biotechnol; Rozners, JACS). These modifications can be done in-lab using commercially available kits ...surprisingly well-tolerated by the Cas9 protein and can increase on-target efficiency. In the same vein, ..., locked nucleic acids or bridged nucleic acids can also be introduced in the gRNA. These modifications... 20-bp targeting region of the gRNA. These gRNAs can be cleaved and rendered useless after less than a... the gRNA is released and the CRISPR-Cas9 system can initiate targeted editing events. Both technologies...There are many suitable dyes to choose from and you can opt to do-it-yourself with a kit or purchase it commercially...

...Overexpression methods flood the cell with protein, which can both interfere with a cell’s normal function and ...to ensure the edited cells are behaving normally can be prohibitively expensive for many labs. We’ve created...information and images generated from these cell lines can be found in the Cell Catalog on our data portal. ...components to physically enter the cells so they can go to work. Figure 3: Inducing the double ...Cell Catalog on the Allen Cell Explorer, where you can also find the certificate of analysis provided with... The editing strategy and design described above can be used to create similar donor plasmids for introducing...

...is highly flexible and can accommodate any genetic elements of interest. It can thus be tailored to address... Alan Wong. The complexity of biological systems can hinder our attempts to study and engineer them, but...specific genetic perturbations. The ease with which one can design and synthesize CRISPR guide RNAs (gRNAs) for...provides a means to create barcoded gRNA libraries that can be used to combinatorially modify the genome, screen...different gRNAs in each plasmid. Each gRNA combination can be tracked and quantitatively analyzed by sequencing... With a list of gRNA targeting sequences, you can then readily generate your barcoded gRNA library ...interrogate and understand complex biological systems. He can be contacted at [email protected].   References 1. Cheng...

...that can impact reproduction include embryonic lethality, a short lifespan, or infertility and can arise...modeling blog series, we covered techniques that can be used to introduce genetic modifications into mouse...growing colony of genetically engineered mice, what can you do? In this post, we’ll cover why and how to ...transgene in order to see an effect. Hemizygous mice can be included in breeding strategies identical to those...mutation at one, but not both, of the alleles. You can cross homozygous or heterozygous mice from your F1... of integration of the transgene. These problems can be mitigated in different ways. Prior to engineering...transgene. In existing strains of mice, the transgene can be maintained on a hemizygous or heterozygous background...

... subtype in the brain that can either activate or inhibit neurons. You can find information more about...inflammation, and neurotransmission. These receptors can be activated by an array of different ligands, including...signaling  So with the hundreds of GPCRs out there, how can scientists decipher the signaling pathways from ligand...the transcription factor from the receptor, which can then activate expression of a luciferase reporter...potential to discover new ligands for unknown GPCRs. You can find the entire PRESTO-TANGO kit at Addgene! TRUPATH...the 16 non-visual heterotrimeric G proteins that can be activated downstream of GPCRs. This collection...subunits of the heterotrimeric G protein complex that can be used to detect G protein activation via bioluminescence...

...The computer scientists can get involved in modelling, the electronics folks can build a robot worm on ...meaning that we can obtain known mutants at $7.00 a strain. Makerspaces like ours can also make accounts...technologies and spaces where interested parties can do research. This interest manifested itself in the...on top of that model, and the biologists can get involved a in variety of aspects from the molecular biology...but that would be useful to our model  so that we can use CRISPR/cas9 to build the mutants, analyse their...intend to focus on motor neuron knockouts so that we can perturb factors related to worm motion. Working on... area, please stop by. And if not, I suspect you can find a makerspace somewhere near you. The place to...

...so much for how bright you can make a reaction but for how sensitive you can make a reaction. NLuc requires...fluorescent proteins (FPs) can be problematic because excitation light can cause phototoxicity, autofluorescence...tools are fantastic examples of technologies that can accelerate your research. The creation of NanoLuc...by external light sources. These external sources can generate autofluorescence and have limited penetration...protein fusion (OgNLuc). Both fluorescent proteins can be excited by Nluc emission and the lab has dubbed...nm – a clear demonstration of why a BRET reporter can improve deep tissue imaging. The constructs for GpNLuc.... The technique of coupling FPs with Luciferases can overcome these problems but has been hampered by ...

...sensitivity, which can be problematic if you are trying to show that your antibody can distinguish between...results so that others in the community can benefit and we can all help improve antibody reproducibility...antibody has been appropriately validated so that you can have confidence in your results. But what is appropriately...that you receive; citing existing validation data can be acceptable. However, it is your responsibility...for the target antigen - Just because an antibody can bind to its target, does not mean that is all it ...represent five experimental strategies researchers can use to show that their antibody performs as expected... types are amenable to genetic manipulation, nor can all genes be easily knocked down or out.   Example...

...lab alumni as you can or schedule video/calls if they are not local. Alumni meetings can be the most informative...lab such a big deal? It’s actually something you CAN choose and it will make a huge difference for your...right and responsibility to choose a lab where you can thrive and do your best work. This post is focused...choosing a lab, but almost all of these guidelines can apply to any workplace or job. It amazes me when ...questions in the sections below. Mentoring relationships can help identify your priorities and needs, but sometimes...informative way of gathering data. Former lab members can give open, revealing information about the culture...opportunities with the lab head. Former lab members can give you a fuller picture. Consider talking to people...

...not all cell types can be labeled, and that while multiple copies of the transgene can integrate into cells...recombination is required before fluorescence can occur. Cre recombinase can be used alongside common, existing ...label an organ or entire organism. Because each cell can have multiple copies of the transgene that will recombine...express the construct but do not undergo recombination can be detected by immunostaining of the truncated YFP...and fluoresce in the same color as the fused XFP can be used to amplify the signal. Imaging endogenous... extremely versatile technology as recombination can be induced in developing fish via Cre protein microinjection...mouse (Snippert et al., 2010), a reporter mouse that can be used to track any cell type when crossed with ...

...nuclease, can be used in conjunction with Cas13 to amplify the signal of a single target. Csm6 can cleave...laboratories. In the case of the COVID-19 pandemic, qPCR can be used to diagnose the presence of SARS-CoV-2 RNA...reporter. The detectable signal from this reporter can be measured and quantified to determine the presence...an RNA guided RNase (Abudayyeh et al 2016). Cas13 can be guided by a single CRISPR RNA (crRNA) to cleave...non-specific ssRNA cleavage. How does SHERLOCK work? Cas13 can be programmed with crRNA to target an ssRNA of interest...recognizes and binds to the programmed sequence, it can promiscuously cleave surrounding ssRNA molecules....speed, ease of use, and portability. This method can be used for an array of applications including clinical...