...Associate Director of the Cancer Program at the Broad Institute of Harvard and MIT. The notion of cancer precision... many tricks up their sleeves and most patients still don’t benefit from this approach. One central bottleneck...We've developed a new plasmid toolkit, the Broad Institute Target Accelerator Plasmid Collection, that we...have been two huge challenges preventing us from testing this hypothesis: There was no large scale, broadly...cellular pathways). It wasn’t clear whether gene-agnostic bioassays could accurately test for molecular ...variant function and cancer phenotypes. Broad Institute Target Accelerator Plasmid Collections To overcome...this first challenge, we’ve developed the Broad Institute Target Accelerator Plasmid Collections, now widely...

...David Root from the Broad Institute and Alex Chavez from the Wyss Institute were instrumental in helping...School, and Jonathan Ting’s lab from the Allen Institute have supported the AAV initiative from the beginning...at Addgene. Our Board helps keep our focus on assisting scientists and accelerating research and discovery...Team has been developing expertise in answering questions on virus and helping provide educational resources...word out about this new resource and providing fantastically useful educational content on viral vectors....internationally and be ready to provide extensive logistics support for export and shipping on dry ice. The...permission from relevant plasmid depositors and their institutions to distribute viral preps derived from plasmids...

...on-target specificity. Design and testing of SpCas9-HF1 Kleinstiver et al and the Joung Lab similarly ..., researchers from the Zhang lab at the Broad Institute, the Joung lab at Harvard Medical School, and ...activity of the Cas9 nuclease itself. Design and testing of eSpCas9 Looking at the structure of the Cas9... Slaymaker et al. made a variety of alanine substitutions throughout the groove in 32 separate Cas9 mutants...multiple genomic locations in HEK cells. Further testing of two of the mutants, SpCas9(K855A) and eSpCas9...interaction sbetween the non-target strand and Cas9, Kleinstiver et al disrupted interactions between Cas9 and...We hope one of you will be able to answer this question very soon! We look forward to seeing how the research...

...students and postdocs broadly – and thus PI’s and institutions in turn. The first is the passing and upcoming...the NIH recommended pay scale – used by many institutions to set postdoc wages. The second event of note...students will pursue this newly available avenue. Interestingly, both of these changes share a common thread...simply being able to categorize all postdocs at institutions, will be challenging. At the same time, there...level, FoR is currently compiling a database of institutional responses to the FLSA ruling. Similarly, from...move in the right direction, the increase notably still does not meet the suggested postdoc salary ($50,...advocating for change: from developing a strategy to distilling a message for the appropriate audience and communicating...

...list of Fragmid destination vectors currently available. Table 1: Fragmid destination vectors Organism...depicted. (Bottom) Schematic depicting available destination vectors for various delivery methods, along with... with compatible inserts. All destination vectors contain an ampicillin resistance cassette and an origin...components you want, simply assemble them into a destination vector via Golden Gate cloning, perform an exonuclease...sequence for every individual assembly. What destination vectors are available? What you’re building ...Organism Delivery Type Destination Vector Mammalian Lentivirus pRDA_512 (RSV-driven; lentiGuide-based...Mammalian Transposon pRDA_757 (Piggybac; constitutive expression) Mammalian Plasmid pRDA_791...

...types of difficult people: Hostile-aggressives, who are usually antagonistic and impolite, Complainers...work with difficult people? Each time I ask this question at my workshops, I get nods from nearly every ...unresponsive clams, who seem to ignore you and your questions, Super-agreeable friendly types, who don’t follow...have time to mentor you, and Micromanagers, who question every detail of your project and work hours. ...example, you might misunderstand your supervisor’s suggestions and take your project in the wrong direction....chance to review what you have discussed and add suggestions if needed. 7) Always follow through on your end...is a difficult person, the above strategies will still be helpful but you will need to be more assertive...

...cells. These properties make lentiviral vectors fantastic options for delivering shRNA, CRISPR/Cas9 components...membrane pores) transfection. These techniques are still used and efficiently transfer plasmid DNA efficiently...way to re-create a functional virus. Indeed, by testing several vectors and packaging cell line combinations...vector. Lentiviral vectors Although retroviruses are still in use, considerable efforts have been devoted to...However, in this vector production system, there is still a potential risk for the generation of replication...however improving the safety of these vectors is still an active area of research due to the possibility...Replication-incompetent lentiviruses with human tropism can still infect the researcher and insert into her/his genome...

...KRAS mutations: diagnostic and clinical implications." The Journal of Molecular Diagnostics 12.4 (2010): ...Hendel et al. 2015). Low detection rates mean the investigator can be relatively certain that their samples...off-target mutations are essential data points for investigators looking to definitively link genotype and phenotype...algorithms, their genome-wide search criteria are not exhaustive. Mismatch tolerance settings are often limited...larger mismatches (e.g. six nucleotides) that may still lead to off-target double-stranded breaks (Tsai ...Programmable, can detect DSBs in live cells Not as senstive as other unbiased methods, high background BLESS... multiplexing) Requires large cell population, senstive to time since cell fixing HTGTS Not reported...

...healthy and diseased states can also provide interesting insights into disease mechanisms and protein ...interest from its production site to its final destination. As an example, most secreted proteins are produced...pathway to their subcellular or extracellular destination. At the end of their life cycle, proteins may...plasmid collection from Sue Jaspersen (Stowers Institute) and Mark Prescott (Monash University). You can...subcellular structure of interest in the Allen Institute for Cell Science Plasmid Collection. You can also...analysis software produces quantifiable results and statistics that can be used to compare between different...direct molecular interaction without further investigation. One fluorescent microscopy method commonly ...

...your poster visitors had a great idea or interesting questions worth discussing with your lab. If you made...conference Take notes during talks. If you have questions during the talk, jot them down for the Q&A immediately... went to, I made it a personal goal to ask one question each day (this may be more or less difficult depending...speaker from the conference if you have additional questions. If you or your lab identifies a potential collaborator... want to look through themselves and then ask questions. Practice your presentation for both instances...and say something like “Let me know if you have questions or if you want me to walk you through the poster...a notebook handy so you can jot down notes or questions from your poster viewers and trade contact information...

...can answer some of the surprisingly technical questions elementary students can ask. It doesn’t just need...some accountants running our math tables!  Be realistic about what you can teach  5-10 year olds in a ...some crowdsourcing on Twitter and got lots of suggestions. Here are just a few resources: Steam Powered...for 8th graders was a little intimidating. Too simplistic and I would insult the students. Too complicated...  The overarching guideline for all of these questions, and for any topic you might present to middle...talking to 8th graders was the broad range of questions I received. I gave my presentation five times ... it, and trying to answer some of their great questions, was immensely gratifying. I came out of the experience...

...Planning Tips! 1. Take suggestions Some of Addgene’s best events have been at the suggestion of an employee,...imagine, it keeps all of us quite busy. Though there still isn't time for goofing off, the company culture ...Candidates who interview at Addgene frequently ask the question, “What’s your favorite part about working at Addgene... Addgene?” Most Addgenies reply with an enthusiastic, “Our company culture! Addgene is the best place ...animation, but the gist is a common answer to this question.  We truly work in a unique environment where,... familial aspect of Addgene. As our size has drastically increased, it would have been a tight squeeze... it was just beginner’s luck, but I’m feeling optimistic for the next tournament). Minus a little rain...

...no surprise that the U.S. Bureau of Labor and Statistics shows that employment of grant writers is expected...professional. Dr. Cook served as a principal investigator for several research grants. One of the benefits...benefits of being a principal investigator is that you are asked to review grant proposals from others. ...career in the grants profession: 1. Build on your existing skills & passion  Skills such as research, collaboration... development experience and to build upon your existing skills.  2. Find your niche Identify and develop...career goals, identify your niche, and answer questions when you’re negotiating a new job or a new contract...credential from the Grant Professional Certification Institute (GPCI). GPCI evaluates a candidate's expertise...

...Frequently asked questions Now let's get into some of the nitty gritty and answer the questions those of us...reagents as outlined by your institution or country. Have any other questions? Feel free to post them in..., and more. Not surprisingly, we receive many questions from scientists all over the world looking for...answers to our most frequently asked lentiviral questions. This useful resource from Addgene Depositor ...quickly check that a construct is functional before investing time and resources into making a stable cell line...is useful in gene therapy applications where a distinct subset of the cellular population needs to be ...aspects of lentiviral transduction along with suggestions on how to lower the risk. Biosafety should always...

...yet powerful way to tag cellular proteins and investigate protein localization, interaction, and expression...reversible photoactivation - and have enabled sophisticated imaging techniques.     Irreversible photoactivation...victoria) GFP (wtGFP) by mutating Threonine 203 to Histidine (T203H) (1). wtGFP normally contains a mixed population...can occur multiple times by activating with two distinct wavelengths of light (see Table 2). The best known...usually preferred for studying single protein characteristics, as the monomeric state is less likely to interfere...super-resolution imaging usually have the following characteristics-high contrast ratios, bright fluorescence, ...conventional FPs for super-resolution imaging, they still do not emit the large number of photons achievable...

...to the other six mentoring questions in future posts. If you have a question about mentoring that you want...Cepko to learn about her mentoring style. Top 7 questions scientists ask me about mentoring In this blog... series, I plan to provide answers to the top questions scientists ask me about mentoring. Here they are...’ – inspire you.” “Also a mentor can give you realistic expectations. (Don't think you can finish that... is a sounding board--a good mentor asks more questions than answers them.” A good mentor does all of...lighthearted, the article does highlight key characteristics of a good mentor: Yoda image courtesy...Check Out Angela Depace's Talk on "Precision and Plasticity in Mentoring" A recent post on the blog “Tenure...

...Resistance can be intrinsic, meaning a basic characteristic that naturally occurs in the bacterium, and...but has become resistant somehow overtime. One fantastic example of intrinsic resistance would be Mycoplasma... very skilled at the delicate art of survival. Existing virtually ubiquitously in the environment, there...infecting bacteria can take 24 hours or more, and testing for resistance often takes an additional 18-24 ...available. For routine infections, like strep throat, testing for antibiotic resistance isn’t commonly performed...infections is so rare. When antibiotic resistance testing is requested clinically, this involves growing ... antibiotic resistance?  The problem with this question is, unless an antibiotic targets an essential,...

...organization also known as external collaborations. Interestingly, the ability to work with others outside one...important as more complex approaches to scientific questions become popular. There are only so many methods... in Strategic Partnership Management, a very interesting and exciting part of my work. However, this is...collaborations. During my graduate studies at Karolinska Institutet I started working with other groups. First within...minutes. Get to know your collaborators from other institutions personally on conference calls and make sure...opportunities so just pick the one that you feel enthusiastic about and get started. You never know where ...collaborating with your neighboring lab, you've met interesting people at conferences and you have successfully...

...publication at Addgene. Walton RT, Christie KA, Whittaker MN, Kleinstiver BP (2020) Unconstrained genome ...upstream of either NGA or NGC PAM sequences (Kleinstiver et al. 2015). From these screens, they identified...off-target activity (Hu et al., 2018).  In 2020, the Kleinstiver lab reported the development of near-PAMless .... Furthermore, non-SpCas9's may have other characteristics that make them more useful than SpCas9. For... in target genes resulting in the formation of “sticky ends” rather than the blunt ends created by Cas9...which makes it easier to target AT-rich genomes (Kleinstiver et al., 2016). Type VI CRISPR systems, which ... plasmids from this publication at Addgene. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng...

..., an individual experiences progressive muscle wasting leading to death at around 30 years of age. Despite...the amount of research conducted on DMD, there is still no good treatment. The dystrophin gene is very large...approach. Since shorter forms of dystrophin can still be functional, exon skipping is a good option for...non-homologous end joining (NHEJ) pathway. It’s estimated that very little dystrophin correction (about ...could make a huge difference in DMD, and it’s estimated that exon skipping therapies would be applicable...experiment, they found the exon-skipped transcript constituted 59% of total dystrophin mRNA, similar to the ...CRISPR therapy closer to the clinic, much work is still needed. First, CRISPR delivery must be optimized...