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...appropriate for your specific research question. Considerations when choosing your Cas nuclease When choosing...experiments, there are three important factors to consider: site accessibility, specificity, and sensitivity...cleaves a site that is not a perfect match to the guide RNA (gRNA) used to target the genomic locus of interest...interest. As these off-target effects can be genome-wide and, in some cases, difficult to determine, the ... off-target cleavage should be an important consideration during any experiment. Sensitivity Cas9 sensitivity...measured by calculating the on-target activity divided by the off-target activity. Figure 2: Specificity...engineered Cas9, SpCas9-NG, which required only the di-nucleotide PAM NG (Nishimasu et al., 2018). The David Liu...

...Madison lab created SRIRACCHA in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA...well. The ratio of RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using...fluorescent reporter. Lastly, once a mutant is identified, the PB transposase can easily and seamlessly...fluorescent protein for assessing CRISPR/Cas9 activity Guide RNA structure and the site in the genome you are...Sander and Joung, 2014). Thus it is essential to identify the best sgRNA to ensure success in your CRISPR...-terminus of mCherry can be targeted by a sgRNA guided Cas9 causing a DSB break. This DSB is repaired ...repaired by NHEJ which depending on the nucleotides inserted or deleted, can result in an in-frame shift of mCherry...

...himself from the idea of starting a company around engineered bacteriophages. He decided to quit his "safety...Operations of Sample6, for advice on how to take an idea developed during graduate school and turn it into...into a biotech company. Transitioning an idea from academia to a startup Mike Koeris and fellow co-founders...co-founders Tim Lu and others first came up with the idea that would evolve into Sample6 as graduate students...using bacteriophages as bacterial therapeutics. This idea was incorporated into Novophage Therapeutics, Inc...that entrepreneurs must clearly communicate their idea at various levels of complexity and abstraction ...people with diverse backgrounds how amazing their idea is. While communication styles in academia and industry...

...structure of the POI would work best. Ideally, structural information can guide this decision. In our experience... This is an important downside of the technology and should always be considered when interpreting results...that is strong evidence that the fusion behaves well. But this is not conclusive evidence that the fusion... any tendency of GFP to homodimerize should be avoided because it will double the size of the protein....or (ii) designed to form an inert, unstructured peptide and therefore consisted of glycines, serines and...from Aequorea victoria GFP sticks out and can be considered a linker (figure 1). In fact, for several FRET...al., 2011).  Although structural information may guide design, it is advisable to generate multiple constructs...

...size, the cytometer provides you with two measurements: forward scatter (FSC) and side scatter (SSC). To... being considered for the analysis. (C) A more liberal gate allows more cells to be considered for the... SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind, the voltage...scattered forward at a low angle (FSC) or to the side at a large angle (SSC). FSC and SSC depend on the...three gates will likely be: the FSC/SSC gate to identify cells, the single cell gate to exclude duplicate...running my flow analysis, I can however clearly identify my lymphocyte population (10.7% of all recorded...potential that can be applied to the photomultiplier inside the cytometer to increase the electric current ...

...and Golgi apparatus and outside the cell, most cysteines are oxidized in disulfide bonds. Reactions also...between a genetically encoded peptide (DogTag) and protein (DogCatcher); ideal for protein loop regions ...child (some of us still do), so we are used to the idea of general interfaces and the freedom to test out...2025). Figure 1: Modular assembly tools, alongside criteria for Click Biology. Created with BioRender.com...Click Chemistry reactions, one component bearing an azide reacts covalently with another component bearing...nucleic acid sequence (Addgene can help with this!). Besides requiring stable covalent reactions, other required...required parameters of the coupling system include: Wide fusion-tolerance — Click Biology tags should be ...

...clinical applications could be considered. It is important to consider the question of broader applicability...underwent repair not from the single-stranded oligonucleotide (ssODN) template supplied by Ma et al., but...allele rather than the supplied single-stranded oligonucleotide (ssODN) template. Embryos also underwent editing...failure of the embryos to undergo editing using the provided ssODN template. In fact, this finding led the ...conducting research in embryos should our society decide that germline editing is permissible in cases of...disease prevention. However, it is important to consider that already-tested CRISPR alternatives are applicable...target. In addition, as on-target editing rates vary widely from gRNA to gRNA, it’s unclear where this study...

...week. Here we will provide an inside look at the steps we take to verify the identity and quality of the...plasmids through Addgene is that we provide an added level of confidence in the plasmids we distribute through...the plasmids we make available and provide some advice on the steps you can take to verify your own plasmids... ends to verify that the sequence assembly and identity of the insert are correct. In other cases, we ...glycerol stock to each of 2 96-well microplates (identical copies, see image above). We then select forward...corresponding glycerol plate on dry ice to the sequencing provider. Each sequencing service will have different requirements...requirements for volume and concentration, and not all providers are equipped to perform Sanger sequencing from...

...to start considering my future path. Talking to Addgenies has helped me get a much better idea of what ...organization beyond my job description.” Our resident bagel provider, source of energy, and conversation master...friends in the Biochemistry major seem to have the idea that if you don’t get your PhD you’re set up for...undergraduate school and studying for the MCAT, she decided that she didn’t want to take it. “I realized if...to her. Addgenie Shreya Vedantam with our resident dinosaur! Read her Instagram feature here. ...scooped, she said. Reflecting on her values, she decided that this “is not [her] view of science. It should...joined an investment club and discovered the business side of science, she stuck with it. Lab Manager Shannon...

...Editing Guide RNAs Download the CRISPR 101 eBook Resources on Addgene.org Read the CRISPR guide Find CRISPR...tools. These papers are chock full of great tips to guide your experimental design, especially the design ...nickase + reverse transcriptase and a prime editing guide RNA (pegRNA). The pegRNA consists of a CRISPR gRNA...successful, although sequences with G/C content outside this range can still be optimized (Anzalone et ...transcriptase template, starting with about 10–16 nucleotides. For longer templates, it is even more important...influence the success of your prime edit. If possible, consider following these suggestions: Edit the PAM along...DNA mismatch repair (MMR) is less efficient at identifying these “bubbles” of mismatched bases, enhancing...

...get an answer, consider asking to avoid unrealistic expectations. And finally, provide feedback. If ... in customer service, I've daydreamed about the ideal customer service interaction - one that gives our...experience is based solely on my time at Addgene, I’m confident that these tips and tricks will apply to any biology-related...Your time is precious. Here are a few things to consider before you commit: Double check to make sure ... it’s not in the best interest of any company to hide information about their products from you, unless...papers that successfully used said plasmids. We provide these references as our way of saying “Hey look...can be a particularly good resource if you are considering using a reagent for “off-label” purposes. When...

...individual gRNA from this single vector) and guide-only (lentiGuide expressing only an gRNA, for use with cells...new model or a new assay or both, and you have no idea what genes are involved. Or you don’t know how well...Or you don’t have enough cells to execute genome-wide perturbations. So, while some might argue that the...the CRISPR kinome library? Thus, we are glad to provide CRISPR libraries targeting the mouse and human ... a well-annotated collection of genes that can provide a useful starting point for developing a model ... libraries are available from Addgene as pools provided in two sets, each with 4 gRNAs per kinase, so ...opposed to the 70,000 or more gRNAs found in genome-wide libraries, and thus can be screened in pooled format...

...with people inside and outside your field. Your colleagues, lab mates, and PI can provide feedback on ...poster – now it’s time to practice your delivery. Consider your audience: Who will be attending the session...audience so that they can digest what you’re saying, provide constructive criticism, and ask thought-provoking... for Cancer Research Annual Meeting, you might consider including more background on how your virus of... points will help you remember what to say and provide your audience with a clear picture of your work...on your technical presentation, but those outside of your field can point out places where you gloss over...you’re standing at the session. Present with confidence Now that you’ve printed your poster and practiced...

...though it has been shown to identify trends correctly (Muhamad, 2011;  Providenti, 2006). Its biggest advantage..., look for the lowest-cost option that provides you confidence in the results you need.  Whichever ...Taqman’s increased sensitivity allows for a more confident determination of copy number, albeit within a ...to a standard curve, and most results can be confidently interpreted as integers. Since each sample is...approach is right for your experiment. You’ll have to consider:  Resources needed Most molecular biology labs...exact copy numbers or just general trends? How confident do you need to be in your determination to move...determine exact copy number and it may not be able to identify trends in cases where the copy number variation...

...’s possible to get an idea about the subcellular structures your protein resides in and potentially which...structures, called organelles. Take a sneak peak inside a cell with the images from the Allen Cell Explorer...transport between healthy and diseased states can also provide interesting insights into disease mechanisms and...in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth review of quantitative colocalization... and well-executed experiments require some consideration of FP properties. It is important to look out...Barbier Lab can be used to fluorescently label a wide variety of gram negative bacteria (West Virginia...Virginia University School of Medicine, 7). Identifying the subcellular localization and composition of cellular...

...out Addgene's western blot protocol and protocol video!  Antibodies The most important part of your western...  Lysis buffer When choosing your lysis buffer, consider the subcellular location of your protein. Proteins...SDS-containing lysis buffer, like RIPA, should be considered if your protein is difficult to solubilize, such... protein of interest is small, you may want to consider a smaller pore size (0.2 µm). Conversely, if your...well for proteins of most sizes and are worth considering if your proteins of interest vary wildly in size...used for multiplexing, and the solutions used to provide the chemilumescence may be light-sensitive. They...size. However, HRP is not compatible with sodium azide, a common microbial agent used in antibody buffers...

... CRISPRi screens to identify essential genes and phage host factors, and identify sequence-specific toxicity... inducible CRISPR/Cas9 activation E. coli genome-wide inhibition library CRISPR knockout libraries for...break at the genomic loci of interest and on either side of the cargo (ex: fluorescent protein gene) on the... donor plasmid cargo are complementary to either side of the double-stranded break in the genomic loci...the drug inducible Cas9 plasmids E. coli genome-wide inhibition library In the last edition of “What’...in CRISPR,” we shared with you an E. coli genome-wide inhibition library from the Chong Zhong lab. Now...Now, we’ve included another E. coli genome-wide inhibition library from David Bikard’s lab. The Bikard ...

...transposons due to changes in coloration of corn kernels. Aside from corn, transposons are in the genomes of many...encode a transposase enzyme.  Class I TEs are considered replicative since every time they jump they make...transposons are used in the lab, the transposase gene is provided in trans so that a gene of interest can be inserted...preferred target site for integration is TA dinucleotides and it leaves behind the CAG DNA footprint from...with next generation sequencing, and analyzed to identify which insertions were positive or negative selected...site preference and host range are important to consider when selecting a transposon for a mutagenesis ...regions, gene disruption is less of a concern. RNA guided transposons insertion One drawback to DNA transposons...

...reagent-grade endangered Martian chicory root, but considering this is a needle in a haystack type of objective...In retrospect I used a way too high Cas9 MOI considering that A549 cells are relatively easy to transduce...news for a screen. Because Addgene viruses are provided at a specific titer, you can better plan your ...more difficult to get them to grow out. The only downside is that it takes a few weeks for single cells ...Using this virus, it’s expected that Cas9 activity, guided by the virally-delivered GFP gRNA, will edit out...products (which could be easier to visualize and identify). If you need/want to put the digestion site in...of the PCR product, you would still be able to identify the cleavage product. 3. The Surveyor kit had ...

...meeting and a means to request funding for events, consider reaching out to it. PTAs have more flexibility...Science Night. There are plenty of places to get ideas for science activities. I did some crowdsourcing...teachers socially and we’d already discussed the idea of my giving a CRISPR presentation to their students...students learned something. Based on the initial guidelines I received from the teachers, I formulated the...schooler himself and b) I had the background to be considered an authority in this area. What is genome engineering...CRISPR work? This section was essentially background slides getting everyone on the same page in terms of the... by running it by the teachers and sharing some slides on Twitter, where I was able to get some great ...