... To better highlight the great content contributed by our bloggers each and every month, we've decided...Cpf1 Update: Comparison to Cas9 and NgAgo As you might expect given the excitement surrounding anything...month is from guest blogger James D. Fessenden at Brigham and Women's Hospital. This post describes how to... our posts if you have any questions or useful insights. If you're interested in blogging for us, fill...

...vectors containing high numbers of gRNAs in one overnight reaction. STAgR is highly customizable as vector...In this quarterly blog series, we’ll highlight a few of the new CRISPR plasmids available at Addgene....

...RNA is highly desirable for extending the complexity of genetic circuits, allowing for tighter spatio-...Illinois at Urbana-Champaign. The PUF proteins include eight modular repeats, each of which binds a consecutive..., I think, are endless.” Abil says the new tool might even find therapeutic application in addition to...she recommends using standard BsaI in place of the high fidelity version (despite what the original paper...

...fortunate that there’s a guide that directs cleavage right before the stop codon!  Figure 3: How to ...guide sequences (arrows) and their PAM (bold highlighted text) and cleavage sites (slashes) are shown....plasmid options for templates - see the example at right using blue flame plasmid pmScarlet_C1! After creating...primers to add T7 promoter-left homology arm and right homology arm to the desired insert (e.g., mScarlet...the main drawback of i-GONAD is that it requires a high-concentration, column-purified DNA template.   ...

...not having any confounding mutations, the risk is high. You are generating a gene therapy product with ...clinical trials. An off-target rate of 5% is far too high when considering human patients and the diseases...measurements at those sites to get an idea of how high the off-target frequency is (more on this below)...sequencing methods. Candidate site sequencing Sites with high sequence similarity to the gRNA can be sequenced...a DNA mutation, off-targets must be quantified slightly differently. For a comprehensive approach, RNA-seq...library screens, the same principle holds true - the higher the number of distinct editing events, the lower...

...fixed sample. Depending on the sample type, this might be frozen or formalin-fixed, paraffin-embedded tissue...Depending on your needs, your sample, and targets, you might decide to do simultaneous IF instead (Figure 1B).... Depending on the goal of your experiment, you might need to quantify the intensity of the different ...spatial relationship between the targets. Or, it might be sufficient to describe or classify the labeled...label a sample that expresses your target(s) at a high level, either naturally or by overexpression, for...similar between the two experiments. If not, it might be that the other antibodies are disrupting the ...

...connection to the hiring manager. They might say yes and they might say no but do not confuse networking...scientist in the academic system has many pluses. I delighted in my graduate school years for allowing me to...personal questions can elicit the most valuable insights you will get from the interaction. Most people...great way to practice for real interviews where you might spend a whole day having to talk with strangers....

... all night was hand-picked (like our plasmid samples!) by Addgenies. If you were there, you might have...plenty of delicious desserts. We enjoyed a video highlighting the changes at Addgene over the last 10 years...Perhaps the most heartfelt portion of the entire night was when Melina and Ken called their parents up ...

...customized to highlight different skills and to get the important information in the right place on the...organization is small, any person at the company might be able to help you. If the organization is large...additioanal advice for the science career search.  You might also enjoy the other posts in the Addgene Blog Scientist...

...and can exhibit recombination levels as high as 1-3%. In light of this finding, we now include guidance...leaky expression. On the right non-homologous scrambled recombination sights lead to reduced reversion...strategy should work, these vectors suffered from high levels of spurious expression.  As such, researchers...weak and may require longer waiting periods and/or higher injection titers to achieve adequate expression...

... homozygous knock-in animals can be obtained in eight days. The only animals on a plate, other than the... delivery, screening, and more. Here are some highlights from the past few years:  Repair template design...improvements from the Mello lab resulted in strikingly high editing efficiencies, obtaining as many as 100 independent...corresponding to the same strand as the PAM produced a higher editing efficiency, while for edits 3’ to the PAM...templates. They found that Cas9 RNPs could be toxic at high concentrations, and editing efficiency improved ...

...proximity to an endogenous gene. Each trap uses a slightly different approach (described in detail below)...for the endogenous gene – its expression levels (high/low) and patterns (tissue/timing) can be monitored...screened. Depending on the marker, traps can be used as high-throughput screens to introduce reporters throughout...the top trap – they don’t require insertion in a highly specific gene region and they don’t disrupt the...Resources References Wood, V., et al., Hidden in plain sight: what remains to be discovered in the eukaryotic...

...yet familiar. The idea that my piecemeal degree might one day foster a meaningful day job only came into...enjoyed a taste of what was to offer, continued straight on to a master's degree in science communication... one day required of me for work, I would have a high level of career satisfaction indeed.   For many ...science. Understanding just how the public utilizes insights gleaned from scientific research is, after all...law to poetry. A research discipline in its own right, science communication reflects a necessary confluence...

...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ... TX-TL system is capable of robust expression of high G+C content genes, as validated through monitoring...CRISPR Plasmids and Resources page. Here are some highlights from the past couple months including several...

...are very valuable biological tools, and with the right safety considerations, they aren't dangerous for...transductions can offer benefits such as tissue-specificity, high efficiency, and can be selected to be either integrating...our viral selections to see if we have one that might work for your next experiment. Using transduction...Answering these questions will help guide you to the right method for your experiment.  A cool feature of viral...virus can sound daunting, but it is easier than you might think. We have protocols available to get you started...

...an advantage to use the cell line and we will highlight some of those applications here.   Virus production...transfect in bulk and produce recombinant proteins at high levels. In a case where clinical applications of...and if your research doesn’t allow for that, HEK might be the cell line for you. Are HEKs for you?   If...you are looking for a human cell line model, HEKs might be a great option! Especially if your research interests...

...these visualization tools is CRISPR-Sirius, the brightest CRISPR-based tool to date for visualizing genomic...chromosomes in living cells, the system requires high-copy chromosome-specific loci in the genome and ...the gRNA target site. In addition to increased brightness, they also found that the signal to noise ratio...its predecessor CRISPRainbow, CRISPR-Sirius is brighter making it suitable for observing low-copy genomic...copy genes. If you want to visualize genomic loci brightly, give CRISPR-Sirius a try. Find CRISPR-Sirius ...

...with a large range of characteristics (color, brightness, photostability, maturation, oligomerization)...choosing a FP, but much of the primary data one might require when making such a decision remains spread...there is a great deal more information that one might like to store about FPs than is currently represented...multi-parametric search queries.  Looking for a bright, monomeric FP published in the last few years with... for me? A Practical Approach to Choosing the B(right)est Fluorescent Protein Choosing Your Fluorescent...

...optimal for experiments requiring high signal to noise ratios, imaging of high frequency glutamate signaling...to create SF-iGluSnFR. This improved variant has higher expression levels in bacteria and produces stronger...properties The improved variants of iGluSnFR3 have higher expression levels and produce stronger fluorescent...Biosensors A Practical Approach to Choosing the B(right)est Fluorescent Protein Visit the Fluorescent Protein...

... be ideal for you. Here, we will chat about who might want to explore this role, how to seek out such ...commitment, planning, and funding – they may not be right or necessary for many scientists! Unless some of...from third parties or your home institute. Discuss rights Do you expect to generate data that will contribute...It is never too early to discuss authorship and rights for anything that could be generated during the...opportunity, but also a really good time! If it sounds right for you, start thinking outside of your current ...

...buck - it grows at higher cell densities than most of its counterparts, yielding higher production power... phaffii in the 1970s with the goal of producing high protein animal feed. Due to increasing prices of...flask volume. What’s even better? K. phaffii is highly efficient at protein secretion, making harvesting...knock-in or knock-out) is essential to your work, you might want to pick a yeast strain best suited to it. Putting...

...grandparents’ medical textbooks by torchlight under the covers. In high school, I went to all the optional...technical jargon that even scientists in neighbouring disciplines might struggle to understand. Why should ...whispered questions from friends and classmates back in high school, and it’s oddly fitting that I’ve ended up...

...figure to the right), we identified R-body mutants that extend at both lower and higher pH. We initially...putting the operon under a strong promoter results in high R-body expression  (Schrallhammer et al 2012). This...environmental conditions. More speculatively, R bodies might endow divergent organisms with new functions. For...understanding of how R bodies assemble and function, we might be able to endow cells with new ways to interact...

...sides of the break, we determined that Cas9 held tightly to three of the four strands of the cleaved duplex... the PAM-proximal side, consistently supported higher frequencies (up to 60%!) of gene editing than single...have shown that this technique supports extremely high frequencies of genome cutting and, when donor DNA...included in the nucleofection reaction, extremely high frequencies of HDR. The detailed protocol for nucleofection...Central PMCID: PMC4383097. 3. Kim, Sojung, et al. "Highly efficient RNA-guided genome editing in human cells...

...In a big lab, the advisor might not be around much and your real boss might be a postdoc – this isn't ...competitive style to spur you to succeed or you might enjoy working closely with others to accomplish ...before you take the position. Am I choosing the right mentor? Make sure your advisors will be good role...candidate to ask lab heads, lab members and lab neighbors during the interview process. What if I want...

... Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...here.  Listen to the hot plasmids episode! New, highly efficient RNAi construct for C. elegans gene knockdown...noroviruses can infect millions annually. Because it is highly contagious, it is essential to contain outbreaks...binding experiments. Potential experiments that one might perform using the purified domains include protein...

...RNA without altering the genome, but Cas13s have higher on-target specificity. New work from Konermann ...package in low-capacity vectors! These small, but mighty type VI-D enzymes are the latest tools in the transcriptome...including Cas13d-specific regulatory proteins and high targeting efficiency, both of which are described...Cas13d deposit here). Konermann et al.: CasRx is a highly potent, specific editor in mammalian cells Not ...characterized Cas13 enzymes, CasRx also displays very high on-target efficiency; where shRNA treatment produced...

...cognitive biases such as motivated reasoning and hindsight bias, we may fool ourselves and our readers into...replications are unavoidable, but can be minimized with high powered research designs. Unanticipated discoveries...become public eventually). It is typically done right before data collection starts, and should include...example, if you hypothesize that growth rates will be higher in treatment versus control, and you think there...of the study, say a fifth time has unpredictably high rates, should be reported as a new discovery that...

...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...close proximity to the GFP-tagged protein (albeit slightly offset due to the nanobody-GFP interaction). The...the AAV capsids (left) and transduction patterns (right).  Both AAV-PHP.eB and the AAV.CAP-B capsids are...mammalian cells. Long sequences traditionally have higher risks of off-target mutations. These plasmids contain...

...to induce high levels of gene activation for both reporter and endogenous genes. It is highly specific ...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...target only requires a small epitope tag (FLAG is eight amino acids, HA is nine), multiple repeats of the...

...hybridoma, you can choose one that produces high-affinity/high-avidity antibodies. The same hybridoma is...drive can be identified by sequencing the heavy and light chains. To circumvent any possible genetic drift...  Figure 2: Changes to the heavy chain or light chain genes within a hybridoma impacts the specificity...validating monoclonal antibodies, their cost is higher than that of polyclonal antibodies. For example...

... visitors with information about images that a sighted reader would glean from the image itself.  As we...impaired readers receive the same information as sighted readers.    Figure 1: An image from AAV...image, we thought it would be most helpful to highlight the technical details of collecting the purified... passage of supernatant through each layer. The right panel illustrates a gradient tube before and after...

...Since its early years, the light microscope has been a powerful tool in almost all disciplines. In biology... cells, has become an established alternative to high-priced commercial devices.     Fig. 2: Workflow...Workflow for open software (left) vs. open hardware (right).    Taking this all together, the essential...open hardware. Upper left panel shows the path of light through a microscope. Lower left panel shows an ...differentiating monocyte with planes and hardware to it. Right panel shows workflow of open hardware.    Conclusion...

...surprise then that a lot of our subject matter comes straight from you, whether you realize it or not! Many ...what basic information someone new to antibodies might need to successfully start using them at the bench...bench, along with questions we thought they might have as they go. The guide was then assigned to our science...deeper expertise of our antibody team to rely on. High-impact content like the antibody guide always gets...sketch a western blot out quickly." Final design (right). Immediate and immense regret of the graphic designer...

...does this actually look like? For a selection, you might put cells into a drug-containing media in which ...scientific question can help determine which is right for you. Screen variations Forward and reverse screens...method you choose to assess traits of interest is highly specific to your scientific question. Things to... assay system include: How specific is it? Is it high throughput? Will it be sensitive enough? No one ...screens Selections sound like a great shortcut, right? Who would ever do a screen when a selection can...

...realized that working in the wet lab wasn’t the right path for me. I began exploring alternative career...track and feel supported. Another team member was highly self-motivated and excelled when given the freedom... made similar transitions can provide you with insights, lessons from their experiences, and practical...perspectives and facilitated a team meeting to highlight their complementary strengths. By encouraging ...

...mixture of products or from incomplete PCR — although higher DNA concentrations are needed for this method to...retain products with single-stranded overhangs (right branch). The mixed PCR and incomplete PCR methods...with two 5′ overhangs, so these approaches require higher concentrations of insert to succeed. 5′ overhangs... showed that a five-piece assembly reaction was highly efficient (~80%) with 40-bp homology regions. Ten-fragment...method also based on homologous recombination. The higher temperature at which Gibson assembly takes place...

...is building the software tools for the right people at the right time. To explain a bit further, a software... a strong background of undergraduate research straight to a lab career at a biotech company, I always...operations software for next-gen sequencing (NGS), and high-throughput screening. While some developers had ...about what a software product manager does, you might be wondering what the day to day looks like. Check...

...sensitivity. Wild-type Cas9 exhibits high on-target activity, but also high off-target activity, which may ...sensitivity is the measure of on-target activity (right). Cas9 nucleases that exhibit enhanced site ...et al., 2018) and found that Sniper-Cas9 showed a high degree of both specificity and sensitivity across...Evolved Cas9 variants with broad PAM compatibility and high DNA specificity." Nature 556.7699 (2018): 57. PubMed...

... you to select cells with high GFP expression. These cells have the highest levels of Cas9 and gRNA expression... which method you use, the procedure will vary slightly.   Two Golden Gate options that are available...expression, and thus the highest frequency of genome editing events. Yamamoto Lab Multiplex CRISPR/Cas9.... “Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.”  Nature Biotechnology...al. “A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and...