...should be easy, right? Yes and no. If you’re just trying to create a knockout, you might be happy targeting...near your insertion site, the corresponding gRNA might be undesirable due to potential off-target sites...they saw that xCas9 3.7 cleaves multiple PAMs at a higher efficiency than SpCas9: NGT (4.5-fold efficiency...genome-wide off-targets, since increasing PAM flexibility might also increase off-target activity. Surprisingly,...generation cytidine base editor architecture (BE3) slightly improves NGG PAM editing efficiency over that ...Evolved Cas9 variants with broad PAM compatibility and high DNA specificity." Nature (2018) doi: 10.1038/nature26155...

...daytime. They mostly stop emerging at night. An incubator with a light cycle makes for very predictable work...temperatures like 18°C, flies take longer to develop. At high temperatures like 30°C, flies develop faster but...When flies first emerge, their bodies will be very light in coloration and can look very plump. On the side...flies. Like Addgene, the Drosophila community is highly sharing-oriented, and there are many resources ...your fly gene using the search box in the upper right hand corner. They have video tutorials under the...useful information on topics such as choosing the right balancer chromosomes to use. If you’re not ready...

...False, width='600', height='189', player_id='31668202421', style='text-align: right;' %} Figure 3. An...data with a 7-day rolling mean gave us a crude insight into the magnitude of the effect COVID-19 had on...there are many new COVID-19 cases being reported (right, red), likely due to quarantining {% video_player...most impacted or least impacted. These results highlight the instability and uncertainty in estimating ...methods for fundamental scientific discovery and high-impact applications.       Alvin Hsu is a graduate...

...a new coordinator for the annual Math & Science Night, I took the reigns. I also volunteered when our ... started coordinating the annual Math & Science Night, a chance for parents and other members of the community...activities. If your school doesn’t have a Math & Science Night (or some equivalent), here are some quick tips to...activities we had for my school’s Math & Science Night. There are plenty of places to get ideas for science...for all of these questions, and for any topic you might present to middle schoolers, is to provide current...again next year. There are many reasons why you might want to reach out to your school community as a ... Elementary for trusting me with Math & Science Night, and a special thanks to Joanne Marks, Jonathan ...

...outperformed another CRISPR library with 6 sgRNAS, highlighting the benefit of sgRNA design in CRISPR libraries...CRISPRi sgRNA library While CRISPRko libraries are highly effective at identifying strong hits in genetic...because it's essential for viability. In addition, high copy number genes may mimic viability genes as they...knock-out library while mitigating toxicity induced by high copy number dsDNA cutting. A Dolcetto library containing...wide CRISPRko, CRISPRi, and CRISPRa libraries are highly effective for probing gene function while using... 1. Doench, John G., et al. "Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation...

...Automation and Screening (SLAS) Electronic Laboratory Neighborhood.   Work/life balance. Is it truly possible to...have one-half of the guilt. You can handle that, right? Big tip: this is an important topic to discuss .... The same with sheets – wash them and put them right back on the bed to save a step! There is more than...situation. Go to church, join a club, meet the neighbors and create a network of support to help with elder... elder care, carpooling, cooking on nights when you can’t get home, babysitting or running errands. Give...very, very fast! I will never forget the time my eight-year-old daughter vehemently reminded her doctor...wait and some things can’t. I kiss my kids good night every evening I am home no matter how busy I am....

...from magnetic beads are equivalent, if not slightly higher, than those of agarose beads, despite the size... method is appropriate for most situations.  A slightly different method is desirable if the antibody ... crystallizable region (Fc) of antibodies with a high affinity (Hjelm, 1972, Bjorck 1984). Protein A, ...beads, break weak protein interactions, and limit high throughput procedures. However, agarose beads are...the protein of interest. The wide availability and high specificity of anti-epitope antibodies make this...Tagged proteins are typically expressed at much higher levels than endogenous proteins. Data obtained ...

...Addgene we've set out to fill this gap by both highlighting that researchers do learn MANY transferable ...the lab and by offering advice on areas where you might need some help. Today: Career Planning Resources...graduate school and beyond is to live in your lab, right? Wrong! While it is clearly a good idea to be diligent...there are different types of conferences that you might attend. Societies, such as the American Society ...contact an email with the relevant info and they just might personally hand your credentials to HR, greatly ... institution’s career center. Alternatively, it might even be a colleague. A mentor does not necessarily...

...such as long or highly structured templates. Cas9 variants PE6e–g often had even higher editing efficiencies...Every few months, we highlight a subset of the new plasmids, antibodies, and viral preps in the repository...the utility of this technique.  By engineering a light-sensitive LOV domain into the TurboID enzyme, Lee...LOV-Turbo becomes active when illuminated with blue light, catalyzing the biotinylation of nearby proteins...Cheah, J.S., et al. (2023). Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal...

...Central PMCID: PMC3150193. 8.Han, Xue, et al. "A high-light sensitivity optical neural silencer: development...in your brain region of interest could be low or highly variable without the behavioral test. Another readout...important in memory, an appropriate behavioral test might be contextual fear conditioning (reviewed in [4]...optogenetics setup (viral expression, efficiency of laser light transmission to the target neurons, appropriate ...mistakes. The easiest way to learn optogenetics is straight through the source - find a lab within your own...Filippo, and Claire Wyart. "Optogenetics: a new enlightenment age for zebrafish neurobiology." Developmental...

...need an expression strain? Protein expression from high-copy number plasmids and powerful promoters will...typically expressed in E. coli engineered to accomodate high protein loads using inducible promoter systems (...to all or most expression strains to accommodate high protein levels including:  ompT: Strains harboring... (DE3) pLysE* Chloramphenicol (pLysE) pLysE has higher T7 lysozyme expression than pLysS; expression vector... (DE3)** Streptomycin and Tetracycline Contains highly active thioredoxin reductase and glutathione reductase...—the difference being that the latter provides tighter control of basal expression. What if I don't see...media such as TB or 2XYT is optimal because of the high cell-density they support; however, minimal media...

... In this quarterly blog series, we’ll highlight a few of the new CRISPR plasmids available at Addgene...recently developed a platform called SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) to detect... this further to provide multiplexed detection, higher sensitivity, and more. Read the paper in Science...developed dual-nuclease Cas9-Cas9 chimeras with higher target site activity and more predictable and precise...inhibitor, with the LOV2 photosensor from Avena sativa, light-mediated genome editing is now possible. Learn ...

...institutions of higher education nonprofit entities related to or affiliated with institutions of higher education...reserved for individuals with a US Master’s degree or higher degree. These numerically limited visas are referred...theoretical and practical application of a body of highly specialized knowledge in a field of human endeavor...the cap if the applicant works at institutions of higher education, a related or affiliated nonprofit entity...interest waiver criteria however the EB-1A requires a higher standard of proving that you are one of the top...showcases (not usually applicable to scientists). High Salary –not relevant to most postdocs. Commercial...circulation) in the field EB-1Bs historically have a high rate of approvals with USCIS due to the fact that...

...canonical base editing window, base editing has higher efficiency and fewer indels than prime editing....However, the efficiency of Cas9 cleavage is relatively high while the efficiency of HDR is relatively low, meaning...varies widely — it still almost always results in higher ratios of desired edits to indel byproducts than...average 2.3- to 5.1-fold (though up to 45-fold) higher across different genomic sites compared to PE1 ...process of double nicking increases indel formation slightly. Designing the sgRNA with a spacer that only binds...asked. Hopefully this table helps you keep them straight! Innovation Purpose PE2 PE3 PE4 ...PE6 PE7 Pentamutant reverse transcriptase Higher efficiency ✓ ✓ ✓ ✓ ✓   ✓ Second...

...Proximity labelling relies on the labelling of neighboring proteins within a cell with biotin. Biotin, is...avidin coated purification techniques. Due to the high affinity of biotin for avidin, proximity labelling...promiscuous biotin ligase that can efficiently label neighboring proteins The first biotin based proximity labelling...from the ligase and bind to exposed lysines on neighboring proteins. Thus to capture protein complexes, ...electron-rich amino acids such as tyrosine on neighboring proteins resulting in their biotinylation. The...The advantage of APEX is its speed, labeling neighboring proteins in minutes rather than hours like BioID...sensitivity. Sensitivity could be increased with higher expression levels but for many fusion proteins ...

...some of these plastics are high grade and can be washed and reused. This might sound risky in terms of contamination... in a high-throughput and efficient way? Grenova, for example, offers automated, benchtop, high throughput...are an accepted part of lab culture, but with the right incentives (for example, by grant agencies), we ...behavior and innovate solutions and practices to meet higher environmental standards. Find some easy action ...

...Scientist I/II. Promotions don’t happen overnight though, and it might take longer to advance without a PhD... few questions to help you decide if a master’s might be for you. 1. Are you interested in master’s-...with a PhD, a master’s is less specialized so it might lend itself more readily to a switch. 4. Do you ...the bench. So if you enjoy wielding a pipette, it might make sense to earn a master’s instead of a PhD. ...good fit with your interests and career goals. You might still decide to earn a PhD, but if I had to do grad...

...contains a region with a high amount of guanine and cytosine nucleotides, we might need to send it for Sanger...to confirm the sequence of a plasmid and we’ll highlight some of the new features available on our website...our first clue about where the gene of interest might be located. By easily using BLAST to check the translated...repetitive region is present in the plasmid, we might need to perform a restriction digest to confirm ...

...get to where I was. It was really luck of right place, right time, and having a good interview experience...educators, mostly elementary school, middle school, and high school.  I’m the only science person of the bunch...plans was paved with lots of writing, hard work, a slight detour, and a bit of luck. This journey started... editing These teaching experiences definitely weighed in Caitlin’s post-grad plans, but in the end, writing...services. “I was working during the day and editing at night. It was not the easiest 6 months. But it gave me...

...raging night, With strong heats full, our souls ignite, When all seems lost in the War of Light, Look ...After three kids, I had put on typical middle age weight and “exercise” consisted of taking walks in the...career as Senior Director of Biology, is helping us fight against FOP in two ways. First, Addgene is distributing... their plasmids with Addgene and join us in the fight against FOP. Second, Addgene will be hosting the...                                    -for HOPE burns bright!"   Many thanks to our guest blogger Kurt Swanson...

... your professional brand. It offers colleagues insight into your interests and will determine how you ...Advice from principal investigators Tom Ellis highlights the importance of keeping up-to-date with the...project that aligns with your dreams. He also highlights the importance of combining “new discoveries ...and mentors will help elucidate which project is right for you.   Acknowledgments Thank you to Dr. Tom...

... functions using slightly different Cas proteins and PAM sequences. A total of eight evolutionarily conserved...safeguard against bacteriophages did not come to light until 2007. Initial experiments exposed S. thermophilus...undiscovered viral anti-CRISPR genes. These data highlight an interesting "arms race" between phages and ... to work diligently and creatively to deliver a highly specific, programmable platform well-suited for...

... areas, including one hot spring requiring an overnight hike in. Navigating challenges: collecting and...and processing environmental samples Since the high temperatures and unstable ground around these hot springs...spring. Hiking all day and processing samples by night was fun but also very tiring. It feels a bit strange...gloves at a campsite. And it was exciting – one night while trying to process samples at the picnic table...using microfluidic parallelization to sequence this highly complex hot spring sample. The sample was separated... sequencing, allowing for lower complexity with higher throughput and rigorous assembly. There, they found...

...Fighting with antibodies to produce immunohistochemistry images that are crisp, bright, and lacking in...find high levels of background staining by the secondary, then the following three strategies might help...strategies for mitigating this issue, and we’ve highlighted a few below.  What’s the problem?   First, why...

...well-organized, documented, and easy to find (though we might need to see it to believe it.) As you browse the...epitope that was previously hidden, an antibody might suddenly work that didn't before. This is most often...antibody to bind to a previously hidden epitope (right). Created withBioRender.com.   Biological Processes...it is attached to a cell membrane.  Picking the Right Antibody  It can get even more complex if you’re...configuration. So how do you ensure that you have the right antibody for the protein as it exists in your assay...how the available epitopes in any given protein might change depending on the conditions and the assay...

...very high level of independence, such as museums, YouTube channels and podcasts, science pub nights, and...plumber, a physicist, an ambassador to Spain, and a high school student would all be defined as laypeople...comes to molecular biology.  Some lay people have a high amount of knowledge or interest in STEM subjects...it would be safe to assume that they both have a higher level of science knowledge than average and are...scientist underrepresented minorities in your local high school who are also students in AP biology classes...

...drastic shift won’t happen overnight, but there are a few reasons why you might choose NgAgo over CRISPR...TtAgo-mediated cleavage in mammalian cells, likely due to high temperature requirements. To solve this problem,...preclude future HDR, which could lower HDR efficiency. Higher sensitivity to off-targets More research is needed...other small nuclear DNAs from binding NgAgo, it might make it difficult to use NgAgo to disrupt multiple...Addgene Blog Read About Cas9 Homolog Cpf1 Check Out High Fidelty Cas9 Mutants Browse All Genome Engineering...

...explains, while these Twitch sensors are always bright, they change color as they shift from "off" to ...They identified a toadfish TnC variant which had a high-affinity for calcium binding and developed a version...see tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes,...and acceptor domains. He says, these new colors might now enable observation of the interaction between...Addgene? Check out our Hot Plasmid page, where we highlight some of the popular & newly available plasmids...

... sections of scientific articles (gasp). To my delight, not only was I able to make the edits without ...all about the MOI. In retrospect I used a way too high Cas9 MOI considering that A549 cells are relatively...robust cell line. If your cells are sensitive, it might be more difficult to get them to grow out. The only...presence of the band, you could run another gel that highlights the important results (for example, if you needed...a human cell line with a gene knockout was straightforward, and is a testament to how far genome engineering...David Root and is described in Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation...

...butterflies in your stomach, the virtual approach might be less stressful. For both conferences, there was...Bachle. (For presenters, there’s also less stage fright!)And depending on the conference, the audience ...can upvote a question and the host will see the highly voted questions,” says Zhang. “Usually the moderator...moderator starts by asking the speaker the highly upvoted question.”If the Q&A session ran out of time, ...for Everyone.  While this type of interaction might not work when you are giving a research talk, there...difficult in the virtual format than in-person. You might miss out on the short chats between sessions or ...