9 New Year Resolutions for Grad Students and Postdocs (and tools to help keep them)

By Gaia Vasiliver-Shamis, PhD

Let’s face it, reality is that our new year resolutions usually don’t last past January…while the world wide web is full with “science-backed” ways to keep your new year resolutions, it’s almost had the same number of articles explaining you why new year resolutions don’t stick. But let’s stay positive on New Year evening and use the latest behavioral research (from 4 days ago!) showing that if you want your resolutions to stick you should ask yourself a question rather than make a statement. For example, don’t say “I will exercise” but ask yourself: “Will I exercise? Yes or No?” – the answer should clearly be yes. So in this spirit here are 9 New Year questions every graduate student/ postdoc should have for a successful 2016!

Let’s start with scientific-related resolutions…. I mean questions.

 

  1. Will I finish my paper?

Publications are the token of productivity in the research realm so no matter what your next step may be, you should show that you have been productive. Some struggle because they think they need to get one more experiment done before they start writing…this one big experiment that will turn your work into a Nature paper. While this might be true, this is not the case for most of us. To finish your paper, you actually need to start it! Start with summarizing what you have thus far by creating a sketch of your figures. This will force you to think what is the story you are trying to tell in your paper; this will help you identify the “holes” or the missing experiments in your story. Once you identify those, discuss it with your mentor and make an action plan with some deadlines for the missing experiments and not less importantly for writing!

If you are a visual person or just love “to do” lists, I highly recommend you try Trello – it’s a great, free, tool to help you organize your plans and projects in one fun dashboard (and between us, there is something extremely satisfying by dragging an item to the “done” list).

 

  1. Will I present at a meeting?

I am sometime shocked to discover that some trainees barely attend scientific meetings. Attending conferences is a bundle of important opportunities. Beyond the obvious of learning about the latest research in your field; it’s an opportunity for you to present, get an award (travel award, best poster etc.) and not less importantly network. Yes, I know…networking…it’s so sleazy….so let me rephrase: you will meet new people. Depending on the conference and its size, you will get a chance to interact with top researchers, editors in journals you wish to publish in and scientists from industry and other young scientists like you!

Now, this is something you should plan for, especially if you need to apply for a travel grant so plan early. Nature has a list directory and the myriad of events can be overwhelming. If you’re new to the research business just ask your mentor and peers which conferences they usually go to and recommend.

 

  1. Will I apply for a grant?

Getting funded not only shows that your research is solid and promising, but it also speaks greatly to your written communication skills. It does not matter whether you want to stay in academia or not, having a grant you written get funded will look great on your CV/ resume. Take advantage of any grant tutorials or clubs you may have at your university/ institute and apply even for a small grant. Not sure where to find a grant you can apply to? Try this list from Science Careers.

 

Moving on to career-related resolutions, the arching question you should ask yourself is “will I make the time to take care of my own career?” and the answer should be “hell yeh!”. I’d also like to stress that the following questions hold true whether you are planning for an academic or non-academic career path.

 

  1. Will I attend an event from the Graduates/ Postdoc Affairs Office?

First, if you are not already aware of it existence – find out whether there is a postdoc office or graduates affairs office and what kind of events they offer and make sure you receive their emails.

Once you are in the loop of what’s going on in your institute be sure to attend their events. Whether it’s a CV seminar, career panel or what not – make the time to attend this. I know life happens and experiment go wrong but you should block this time on your calendar for YOU! Taking 2 hours a week to attend a workshop will not stall your research, seriously!

 

  1. Will I intentionally meet new people? (aka the networking more resolution)

I can’t stress this enough! While networking is a pretty dreaded concept for some people (and if you’re one of those people be sure to read “Networking for people who hate networking” by Devora Zack), try to approach it as meeting new interesting people and building relationships. Use LinkedIn and your existing network to identify other professionals you can talk with, reconnect with older or dormant connections or simply join your grad students/ postdocs association. My favorite posts about this topic were titled “Cold emails and hot coffee”. This four –part series in Science Careers shows how you can advance your career in a few hours a week and offers practical tips. Also, if you’d like to stay organize tracking who you talked with and what about, use MyIDP or Evernote.

 

  1. Will I keep my CV/ resume updated?

This is a good habit to form for your professional life in general. Always keep a “kitchen sink” CV/ resume where you add everything you’ve done. I know it’s easy to remember to add a published paper to your CV but you may forget being a member on a committee or writing a piece for the student newspaper or giving a talk so it’s a good practice to add those as soon as you’re done. Also, don’t forget to include any metrics (because sometimes these are easily forgotten).  I’d suggest having your “kitchen sink” CV/ resume as a Google doc so then it’s available for you anytime on any device so you’ll have no excuses (plus you’ll have a backup)!

 

  1. Will I create my career “wish list”?

If you are looking for opportunities beyond academia, you should have 2 lists one for 2-3 career paths of interest and one for companies you’d like to work for. If you’re interested in the academic path, you should have your list of universities/ institutions you are interested in. Once you completed your list, go to question/ resolution #5 and make sure your meet people working in careers you’re interested and/ or people working in the companies/ universities on your wish list.  Since networking is about building relationships – the earlier you start – the better!

 

  1. Will I learn something new?

If you were not into learning new things, you probably wouldn’t have taken the research path right? Whether it’s a new technique in the lab, learning R (which is super valuable on the job market these days) or just expanding your horizons – there are multiple ways for you to learn something new in 2016 and it doesn’t have to cost you a dime! The Muse had a couple of posts with links to FREE online courses in programming, finance, digital marketing and much more, here are the links for 45 courses and 43 Career-advancing courses, you can finish the listed courses in 10 weeks or less. Now who wants to start 2016 smarter?

 

  1. Will I gain a new skill (or develop an existing one)?

You have opportunities both inside and outside the lab to gain/ develop different skills such as leadership, teaching, organizing and more. You should proactively seek opportunities to gain the skill(s) you’re interested in having. Mentoring is a skill that can be easily be acquired when you’re in the lab, if your PI haven’t assigned you someone already, express your interest to her in mentoring an undergrad student or a grad student if you’re a postdoc already. For leadership and organizational skills join the student/ postdoc organization and be active. And if it’s you’re written communication skills you’re looking to strengthen – the Scizzle blog is always on the lookout for talented writers, so drop us an email if you’re interested.

I know, these are some very serious resolutions and it may seem overwhelming at first. A known way to set goals and achieve them is 1) to write them down and 2) have them be SMART goals (Specific, Measurable, Action-oriented, Realistic and Time-bound) and you can learn more about how to set them here. If this is not enough find an accountability buddy, it can be your friend, your spouse or the career person in your grad student/ postdoc affairs office.

Now I know some of you must be thinking “I don’t have time to do my research AND all of this!”. I’d urge you to put your own career and success and a top priority. Taking even a couple hours out of the 168 hrs you have in a week to advance yourself is very doable. Not convinced? Try  Toggl that allows you to track time based on tasks/ projects or use it to time your career-related activities or find out how much time you really spend on pointless browsing by using Rescue Time.

And if all this is too stressful for you, try this fun website called Pixel Thoughts, it will help put things in perspective and calm you down in 60 seconds. After all, mindfulness meditation is the like new non-GMO (though it’s actually a trend that is based in solid science).

I hope you find this post useful and I wish you a very happy and successful new year!

 

 

New year’s resolution #1: planning efficiently the end of your PhD!

 

By Sophie Balmer, PhD

 

2016 is already knocking at the door and with it comes new resolutions. If one of yours is to defend your PhD in 2016, then you are reaching your goal and that is great! But do you know the various challenges you will face during the process?

 

I started planning the end of my PhD roughly a year before I defended. I needed to know exactly how things would go and I had a whole plan for it. However, as for the rest of my PhD, not everything went according to the plan… On the other hand, it taught me very valuable lessons that could be useful to others. Here are 10 short advices to help you succeed!

 

Know what you need to prepare for the submission of your thesis

The first thing to do is to read and understand the guidelines established by your graduate school. Research which paperwork you have to fill in, how many signatures you will need and most importantly the deadlines to respect. It is always better to prepare everything in advance instead of running from one office to the other on the last day before submission.

To write your thesis, inform yourself on the format required and try to get an idea of approximately how many pages each section is. It is also useful to know if you have to include everything you have done or if should you just include your published work. And if you are still confused, ask your advisor for the thesis of his/her previous graduate student and read them. You will find all the information you might need there.

 

Schedule your thesis defense as soon as possible

Some graduate schools will ask you to have one last committee meeting to get the authorization of your committee members to defend. From there, you will have several months to focus your energy on preparing for the D-day.

 

Plan everything in advance

Got the date? Great, it allows you to plan these few months accordingly. You should set aside a significant amount of time to write your thesis, including the time needed to get corrections from your peers or advisors. The best would be to plan on finishing everything about 1-2 weeks before the deadline dictated by your graduate school. These weeks will be very useful if you need to finish figures, make a few last-minute corrections or read everything one last time.

 

Don’t be too ambitious!

One thing about planning ahead is that sometimes we tend to overbook ourselves. Be realistic, start with small goals and then the bulk of your writing time can be more intense. Also, if you know you do not work well in the morning or at night, plan something easier for those hours. Moreover, I would advocate for days off where you really take some time to think about other things and do something not related to your work. You will come back refreshed and less frustrated of spending all day writing.

 

Improve your efficiency

There are many ways to improve efficiency. From taking a nap in the middle of the day to going for a short walk, it is clear that taking breaks is really important to be efficient. In the world of technologies, there are now apps to help our effectiveness. I was recently advised to use the “pomodoro technique”: focus for 25 minutes on one task only and take a 5-minutes break. Then repeat this cycle 3 times and take a 15-minutes break. As always, there is an app for that! At first, 25 minutes feels quite short but the more cycles you accomplish, you start realizing that it is a great way to concentrate on each task. I had achieved so much by the end of the day that it is now part of my routine. The key is to do absolutely nothing else during these 25 minutes. Your friend’s messages can probably wait until your timer goes off!

 

Get other people’s opinion on your work!

Do not forget to give your thesis to read to other people to get their advices on how to improve your thesis, especially your advisor (who should read it entirely if possible). However, make sure you give your reviewers enough time to go through everything you gave them. It is better to give different parts of your thesis to several people to split the work among them and then to your advisor at the end, who will also have less work to do.

Don’t wait until the last minute!

Once your thesis is submitted, start preparing your thesis presentation in advance and allow others to look at it or rehearse with them! It seems scary and some of us do not like to hear criticisms but other people will ultimately see your defense presentation on the D-day so better to be prepared. Maybe you could ask your colleagues and then have someone that doesn’t know your work (or at least not in details) look at it with you, they will spot all the information that is missing for a broader audience.

Stick to your plan!

You spent some time planning the end of your PhD for a reason. Do not delay everything to finish that last experiment because research projects truly never end…

 

Think ahead of your next move!

Transitioning to your next career step is not easy, so start exploring early the various post-docs opportunities in academia or industry as well as other career paths. There are many for PhDs and if you doubt it, there are several articles on this blog that can help ease the transition from your PhD laboratory to your next venture.

 

One last thing…

Enjoy it as much as you can! I know it sounds silly, but once you get started, your brain will generate tons of ideas, this is the time you can take to reflect on everything you have done and what the next steps would be if you were to continue on this project! And despite the amount of stress it generated, this is by far the most fun I have had during my PhD!

Scizzle’s CRISPRmas Science Gift Guide

By Sally Burn and Gaia Vasiliver-Shamis

Holmium Holmium Holmium (Ho Ho Ho)! Scizzle is in a festive mood and ready to deliver a science-themed treat down your chimney – that’s right, it’s the annual Scizzle festive gift guide! This year CRISPR/Cas was top of many santas’ experimental wishlists (still not sure what CRISPR is all about? Check out our recent article on it here), so in honor of this awesome genome editing tool we’ve renamed this year’s edition “Scizzle’s CRISPRmas Science Gift Guide”! Enjoy and make sure to take a look back at our 2013 and 2014 gift guides should you need any further inspiration.

 

Gifts for Her

Struggling to find a gift for a science-loving lady? Let her wear her love for science with our handpicked selection of jewelry, clothing, and accessories:

 

Science Trinkets

For the general science-phile, look no further than this classic silver “science” name necklace – basically what Carrie Bradshaw would have worn had she been as passionate about Erlenmeyers as she was about Manolo Blahniks. Need something more niche? For DNA aficionadas, may we suggest the “beads on a string” model of chromatin structure – captured in necklace form. For the evolutionary biologist, this phylogenetic tree necklace ticks all the boxes. Or, for the neuroscientist, how about this snappy serotonin bracelet?

 

Cretaceous Clogs and Stormtrooping Shoes

What’s that you say, you’re looking specifically for a pair of glamorous dinosaur-themed shoes? Here you go then: feast your eyes on these gold glitter T-Rex shoes. Alternatively, if you are looking to splash out on a big bucks gift, treat her to a pair of these awesome Star Wars “The Empire Struts Back Bootie”.

 

Geek Chic

Moving on to apparel, this rocket-adorned “science” t-shirt is both stylish and affordable. Pair it with the science infinity scarf and the fabulous Women of Science Skirt – featuring the 18th century physicist Laura Bassi and chemist Marie-Anne Pierette Paulze – for a complete (if slightly bonkers) outfit.

 

Gifts for Him

Science-loving gentlemen can also expect a treat in their stocking this year with one of these unique and affordable gifts:

 

Science Shirts

Does your man want everyone to know he’s down with Chuck D? No, not the Public Enemy MC, but good old Charlie Darwin. If so, then you should naturally select (geddit?) this “Darwin is my Homeboy” t-shirt. Or perhaps his ultimate science hero is astronaut and botanist Mark Watney, AKA the stranded survivor from blockbuster movie and book The Martian, who uttered the fantastic Neil deGrasse Tyson-endorsed line: “In the face of overwhelming odds, I’m left with only one option, I’m gonna have to science the shit out of this.” Let the world know that he also intends to science the proverbial poop out of this, with an “I am gonna have to science the shit out of this” t-shirt.

 

My Chemical Bromance

Chemistry nerds will go wild for both this periodic table brass cuff and festive-themed bowtie. Complete their outfit with a testosterone molecule belt buckle and testosterone cufflinks.

 

Festive Feet

Offer to clad his stinking man feet in one of these fabulous items of footwear. First up, Absolute Socks sell a range of spiffing science-themed socks – because what’s Christmas without receiving socks? Next, choose from one of these two awesome pairs of bespoke Converse All Stars, featuring either Nikola Tesla or Albert Einstein. Finally, take his indoor footwear to the next level with a pair of Chewbacca slippers.

 

Gifts for the Home

Science-ify your loved one’s home with one of these inspired decorative pieces:

 

Vintage science posters

Your loved one misses the lab when they’re at home? Or just have an admiration for vintage lab equipment? Then consider ordering this set of 4 VINTAGE science posters!

 

Astronaut Bed Sheets

Your kid wants to be an astronaut when she grows up? Great, make her astronaut dreams even dreamier with this super cool Snurk astronaut Duvet cover.

 

Test Tube Chandelier

Fancy that! Light up the house with this hand-made test tube chandelier! Not only it was inspired by Marie Curie, the test tubes are also detachable so you can rearrange them just for fun or grab one if you ran out of clean tubes to test the O.D of your culture.

 

Science Pun Coasters

Keep your table surface clean with these adorable coasters that will make you wish you could have coffee inside the lab. These coasters feature your favorite scientists and even better have some hilarious puns like “we are two peas in a pod” for Mendel or “you’re radiant” for Marie Curie, ‘cause hey, we all need some sense of humor when we’re having coffee.

 

Your DNA on a canvas

Forget the Andy Warhol colorful self-portrait, personalized art just got better! What better way to show someone you love them than by creating a unique masterpiece from their own DNA, and it doesn’t just come in ethidium bromide, you can choose between 16 different colors!

 

Periodic Shower Curtain

Your darling spends a lot of time in the shower washing away the elements with some H2O? Make it a meaningful daily experience and brush up on your chemistry with this lovely shower curtain.

 

I’ve got a PhD

Need a graduation/holidays gift or maybe give the PhD in your life a way to subtly show off they are a smarty pants with this “I’ve got a PhD” mug.

 

Star Wars Gadgets

This guide would be incomplete if we wouldn’t have at least one Star Wars gift idea. Well, we have 75 ideas for you! In this link you’ll find anything from Boba Fett ice cubes to a Darth Vader porch light cover to lightsaber earrings! And did we mention the Death Star cookie jar? This is clearly for the most serious fans!

 

Petri Dish Ornaments

Sharing the holidays with your loved ones? And by loved ones we mean the million of germs you have on and in your body. Well, who said microbes can’t spread the holidays cheer? Add a scientific flare to your tree with these colorful, pretty petri dish ornaments!

 

Science-tastic Stocking Stuffers

Whether you’re playing secret Santa in the lab or want to nurture your science-phile on a smaller scale, the gift ideas below have got you (and your budget) covered!

 

Spuds Clock

Save your loved one either carbs or batteries, because nothing says green energy like a digital clock powered by potatoes!

 

Beaker cookie cutter

This one is for the bakers who loves makers! A 3D printed beaker shaped cookie cutter. Just think about all the crazy fun cookie decorations you can then do!

 

Biology Buttons/Magnets

These magnets are just TOO CUTE! They’re perfect for home or to bring up the morale in your lab or the fridges/freezer-filled hallway on your floor. These adorable illustrations of microbiology, anatomy, cells, DNA or atoms will put a smile on your face!

 

3D Water Bear

Get a 3D model of one of nature’s toughest, yet cutest, creatures – the tardigrade (aka water bear). Since it’s kind of hard to grow them as a pet, this 3D model may suffice.

 

Molecules Memory Game

Strengthen your neuron connections and chemistry knowledge and bring game night to a whole new intellectual level with the molecule memory game!

Leaving Your Mark on the World

By Danielle Gerhard

 

The idea that transgenerational inheritance of salient life experiences exists has only recently entered the world of experimental research. French scientist Jean-Baptiste Lamarck proposed the idea that acquired traits throughout an organism’s life could be passed along to offspring. This theory of inheritance was originally disregarded in favor of Mendelian genetics, or the inheritance of phenotypic traits isn’t a blending of the traits but instead a specific combination of alleles to form a unique gene encoding the phenotypic trait. However, inheritance is much more complicated than either theory allows for. While Lamarckian inheritance has largely been negated by modern genetics, recent findings in the field of genetics have caused some to revisit l’influence des circonstances, or, the influence of circumstances.

 

Over the past decade, efforts have shifted towards understanding the mechanisms underlying the non-Mendelian inheritance of experience-dependent information. While still conserving most of the rules of Mendelian inheritance, new discoveries like epigenetics and prions challenge the central dogma of molecular biology. Epigenetics is the study of heritable changes in gene activity as a result of environmental factors. These changes do not affect DNA sequences directly but instead impact processes that regulate gene activity such as DNA methylation and histone acetylation.

 

Epigenetics has transformed how psychologists approach understanding the development of psychological disorders. The first study to report epigenetic effects on behavior came from the lab of Michael Meany and Moshe Szyf at McGill University in the early 2000s. In a 2004 Nature Neuroscience paper they report differential DNA methylation in pups raised by high licking and grooming mothers compared to pups raised by low licking and grooming mother. Following these initial findings, neuroscientists have begun using epigenetic techniques to better understand how parental life experiences, such as stress and depression, can shape the epigenome of their offspring.

 

Recent research coming out from the lab of Tracy Bale of the University of Pennsylvania has investigated the heritability of behavioral phenotypes. A 2013 Journal of Neuroscience paper found that stressed males went on to produce offspring with blunted hypothalamic pituitary (HPA) axis responsivity. In simpler terms, when the offspring were presented with a brief, stressful event they had a reduction in the production of the stress hormone corticosterone (cortisol in humans), symptomatic of a predisposition to psychopathology. In contrast, an adaptive response to acute stressors is a transient increase in corticosterone that signals a negative feedback loops to subsequently silence the stress response.

 

The other key finding from this prior study is the identification of nine small non-coding RNA sperm microRNAs (miRs) increased in stressed sires. These findings begin to delve into how paternal experience can influence germ cell transmission but does not explain how selective increases in these sperm miRs might effect oocyte development in order to cause the observed phenotypic and hormonal deficits seen in adult offspring.

 

A recent study from the lab published in PNAS builds off of these initial findings to further investigate the mechanisms underlying transgenerational effects of paternal stress. Using the previously identified nine sperm miRs, the researchers performed a multi-miR injection into single-cell mouse zygotes that were introduced into healthy surrogate females. To confirm that all nine of the sperm miRs were required to recapitulate the stress phenotype, another set of single-cell mouse zygotes were microinjected with a single sperm miR. Furthermore, a final set of zygotes received none of the sperm miRs. Following a normal rearing schedule, the adult offspring were briefly exposed to an acute stressor and blood was collected to analyze changes in stress hormones. As hypothesized, male and female adult offspring from the multi-miR group had a blunted stress response relative to both controls.

 

To further investigate potential effects on neural development, the researchers dissected out the paraventricular nucleus (PVN) of the hypothalamus, a region of the brain that has been previously identified by the group to be involved in regulation of the stress response. Using RNA sequencing and gene set enrichment analysis (GSEA) techniques they found a decrease in genes involved in collagen formation and extracellular matrix organization which the authors go on to hypothesize could be modifying cerebral circulation and blood brain barrier integrity.

 

The final experiment in the study examined the postfertilization effects of multi-miR injected zygotes. Specifically, the investigators were interested in the direct, combined effect of the nine identified sperm miRs on stored maternal mRNA. Using a similar design as the initial experiment, the zygote mRNA was collected and amplified 24 hours after miR injection in order to examine differential gene expression. The researchers found that microinjection of the nine sperm miRs reduced stored maternal mRNA of candidate genes.

 

This study is significant as it has never been shown that paternally derived miRs play a regulatory role in zygote miR degradation. In simpler terms, these findings contradict the conventional belief that zygote development is solely maternally driven. Paternal models of transgenerational inheritance of salient life experiences are useful as they avoid confounding maternal influences in development. Studies investigating the effects of paternal drug use, malnutrition, and psychopathology are ongoing.

 

Not only do early life experiences influence the epigenome passed down to offspring but recent work out of the University of Copenhagen suggests that our diet may also have long-lasting, transgenerational effects. A study that will be published in Cell Metabolism next year examined the effects of obesity on the epigenome. They report differential small non-coding RNA expression and DNA methylation of genes involved in central nervous system development in the spermatozoa of obese men compared to lean controls. Before you start feeling guilty about the 15 jelly donuts you ate this morning, there is hope that epigenetics can also work in our favor. The authors present data on obese men who have undergone bariatric surgery-induced weight loss and they show a remodeling of DNA methylation in spermatozoa.

 

Although still a nascent field, epigenetics has promise for better understanding intergenerational transmission of risk to developing a psychopathology or disease. The ultimate goal of treatment is to identify patterns of epigenetic alternations across susceptible or diagnosed individuals and develop agents that aim to modify epigenetic processes responsible for regulating genes of interest. I would argue that it will one day be necessary for epigenetics and pharmacogenetics, another burgeoning field, to come into cahoots with one another to not only identify the epigenetic markers of a disease but to identify the markers on an person by person basis. However, because the fields of epigenetics and pharmacogenetics are still in the early stages, the tools and techniques currently available limit them. As a result, researchers are able to extract correlations in many of their studies but unable to determine potential causality. Therefore, longitudinal, transgenerational studies like those from the labs of Tracy Bale and others are necessary to provide insight into the lability of our epigenome in response to lifelong experiences.

Development On the Fly: An Interview with Dr. Thomas Gregor

By John McLaughlin

 

Thomas Gregor is a biophysicist and Professor at Princeton University. His Laboratory for the Physics of Life uses both Drosophila melanogaster and Dictyostelium discoideum as model systems to understand developmental processes from a physical perspective.

 

Could you briefly describe your educational path from undergraduate to faculty member at Princeton?

TG: As an undergraduate, I studied physics in Geneva, and then moved into theoretical physics and math. I came to Princeton, initially for a theoretical physics PhD; I switched during my time here to theoretical biophysics and then realized that it makes sense to combine this with experiments. I ended up doing a PhD between three complementary disciplines. My main advisor was Bill Bialek, a theoretical physicist. My other two were David Tank, an experimental neuroscientist, and Eric Wieschaus, a fly geneticist. So I had both experiment and theory, from a biological and a physical side. I then went to Tokyo for a brief post-doc, during which I continued in that interface. But I changed model organisms: I switched from a multicellular, embryonic system to looking at populations of single cells [the social amoeba Dictyostelium discoideum]. As a physicist you’re not married to model organisms. When I came back to start my lab at Princeton in 2009, I kept both the fly and the amoeba systems.

 

What is the overall goal of your lab’s research program?

TG: Basically, to find physical principles behind biological phenomena. How can we come up with a larger, principled understanding that goes beyond the molecular details of any one particular system? I mostly look at genetic networks and try to understand their global properties.

 

Do you think the approaches of biologists and physicists are very different, and if so are they complementary?

TG: I’m driven by the physical aspects of things, but I’m also realistic enough to see what can now be done in biological systems, in terms of data collection and what we can test. To find the overlap between them is kind of an art, and I think that’s where I’m trying to come in.

 

Do you have any scientific role models who have shaped how you approach science?

TG: The three that I mentioned: Bialek influenced me in the types of questions that speak to me; Tank had a very thorough experimental approach that taught me how to make real, physics-style measurements; and Wieschaus brought a lot of enthusiasm and knowledge of the system.

 

Your lab has been studying developmental reproducibility and precision, in the patterning of the fly Drosophila melanogaster. In a 2014 paper1, you showed that levels of the anterior determinant bicoid mRNA vary by only ~9% between different embryos. This is a very similar value to the ~10% variation in Bicoid protein levels between embryos, which you demonstrated several years earlier2. So it seems that this reproducibility occurs even at the mRNA level.

TG: Before going into this, the general thought in the field is that things were very noisy initially, and as the developmental path goes along it becomes more refined and things become more precise. This paper basically asked whether the precision is inherited from the mother, or the embryo needs to acquire it. Because the fluctuations in mRNA, from the mother, completely mimic the fluctuations in protein that the zygote expresses, that told us that the mother lays the groundwork, and passes on a very reproducible pattern. So there’s no necessity for a mechanism that reduces fluctuations from the mRNA to the protein level.

 

Continuing on the theme of precision: in a separate paper from the same year3, your lab showed that the wing structure among different adult flies is identical to within less than a single cell width. Did you have any prior expectations going into this study, and did the results surprise you?

TG: Before looking at the wing, I had kind of made up my mind. I had first seen single cell precision in patterning of gene expression boundaries in the embryo. But I also knew that it’s always better to make a measurement first, and it seems that things are much more precise and reproducible in biology than we think, given the idea of “sloppiness” that we have.

 

Do you think that a high level of reproducibility is a general feature of development, or varies widely among different types of species?

TG: It’s a philosophical question in a way, because I haven’t looked. I think what we found in the embryo is not special to the fly; specific mechanisms for getting there might be unique to the fly. For instance, we have also shown in a recent paper from 2013 that transcription is just as noisy in flies as it is in bacteria, hugely noisy. So, physical mechanisms like temporal and spatial averaging seem enough to reduce the high ubiquitous noise that transcription has to the very fine, reproducible patterns that you see in the fly. The specific mechanisms that reduce noise will be very different from species to species, but I think overall the fact that development is precise and reproducible is something we may one day be able to call a principle.

 

If you could make any changes to scientific institutions, such as the current funding system, journal peer review, etc. what would they be?

TG: One thing that might be nice is if we didn’t have to fund graduate students for the first five years of their career; it would be nice to have more streamlined training grants, not only for U.S. but also international graduate students. And so, graduate students wouldn’t have to worry. They should be free to choose a school based on their scientific interests.

For peer review in journals, the problem is the sheer volume of output is becoming so high. One way to keep a peer review system, is either to pay the reviewers money, or to put everything on the bioRxiv [bio archive is a pre-print server for the life sciences] and let some other means determine how to evaluate a paper. I don’t read papers from looking at the top journals’ table of contents every week, I read them because I see people talk about it on Twitter, or my colleagues tell me I should look at that paper, or because I hear about the work in a talk and decide to see what else the guy is doing.

A lot of people are advocating the new metrics – citations, citation rates, H-index – which are so dependent on the particular field and not necessarily a good measure of impact. In 100 years, are we going to look more at those papers than the ones that currently get very few citations? We don’t know. I don’t think the solution is out there yet.

 

Do you have any advice for young scientists – current PhD students or post-doctoral fellows – for being successful in science?

TG: My advice would be to focus on one very impactful finding. If it’s very thorough and good science, it will be seen. Also, nothing comes from nothing. You need to put in the hours if you want to get a job in academia. And I think that’s one of the ways to measure a good scientist, because knowledge in experimental science comes from new, good data.


What are some future goals of your lab’s research?

TG: We’ve been looking at the genetic network in the fly embryo, trying to understand properties of that network. Medium term, we want to incorporate a slightly different angle, which is looking at the link between transcriptional regulation and the 3D architecture of the genome. In the living embryo, we want to look at how individual pieces of DNA interact, and how that influences transcription and eventually patterning. In the longer term, I don’t know yet; I just got tenure, so I need to sit back. Everything is open. That is what’s nice about being a physicist; you’re not married to your biological past so much.

 

In your opinion, what are the most exciting developments happening in biology right now, whether in your own field or elsewhere?

TG: It’s definitely the fact that so many different disciplines have stormed into biology, making it a very multidisciplinary science. I think it makes the life sciences a very vibrant, communal enterprise. Hopefully the next decades will show the fruits of those interactions.

 

This question is asked very often: How do you balance your lab and family life?

TG: When you start thinking about having a family in science, things become much more complicated. Since I’ve had children, my workload went down a lot. My wife is also a scientist, and for her it’s much harder because she’s not yet tenured. As much as people look at the CV and see how many high-profile papers you have, they should also look at it and see your family and life situation. And for women in science, despite all the efforts that have been made, I don’t think we’re there yet.

 

References

[ordered_list style=”decimal”]

  1. Petkova, MD et al. Maternal origins of developmental reproducibility. Current Biology. 2014. 24(11).
  2. Gregor, T et al. Probing the limits to positional information. Cell. 2007. 130(1).
  3. Abouchar, L et al. Fly wing vein patterns have spatial reproducibility of a single cell. J R Soc Interface. 2014. 11(97).

[/ordered_list]

 

 

CRISPR/Cas9: More Than a Genome Editor

By Rebecca Delker, PhD

 

The bacterial defense system, CRISPR/Cas9, made huge waves in the biomedical community when the seemingly simple protein-RNA complex of Type II CRISPR systems was engineered to target DNA in vitro and in complex eukaryotic genomes. The introduction of double-strand breaks using CRISPR/Cas9 in a targeted fashion opened the portal to highly affordable and efficient site-specific genomic editing in cells derived from yeast to man.

 

To get a sense of the impact CRISPR technology has had on biological research, one simply needs to run a search of the number of publications containing CRISPR in the title or abstract over the past handful of years; the results practically scream in your face. From 2012, the year of the proof-of-principle experiment demonstrating the utility of engineered Cas9, to 2015, CRISPR publications rose steadily from a mere 138 (in 2012) to >1000 (at the time of this post). Publications more than doubled between the years of 2012 and 2013, as well as between 2013 and 2014. Prior to the use of CRISPR as a technology, when researchers studied the system for the (very cool) role it plays in bacterial defense, publications-per-year consistently fell below 100. In other words, it’s a big deal.

 

In fact, during my 10 years at the bench I have never witnessed a discovery as transformative as CRISPR/Cas9. Overnight, reverse genetics on organisms whose genomes were not amenable to classical editing techniques became possible. And with the increasing affordability of high-throughput sequencing, manipulation of the genomes of non-model organisms is now feasible. Of course there are imperfections with the technology that require greater understanding to circumvent (specificity, e.g.), but the development of CRISPR as a tool for genomic engineering jolted biological research, fostering advances more accurately measured in leaps rather than steps. These leaps – and those expected to occur in the future – landed the discoverers of CRISPR/Cas9 at the top of the list of predicted recipients of the Nobel Prize in Chemistry; though they didn’t win this year (the award went to researchers of the not-totally-unrelated field of DNA repair), I anticipate that a win lies ahead. The rapid success of CRISPR genome editing has also sparked patent battles and incited public debate over the ethics of applying the technology to human genomes. With all of the media attention, it’s hard not to know about CRISPR.

 

The transformative nature of CRISPR/Cas9 does not, however, end with genome editing; in fact, an even larger realm of innovation appears when you kill the enzymatic activity of Cas9. No longer able to cut DNA, dead Cas9 (dCas9) becomes an incredibly good DNA-binding protein guided to its target by a programmable RNA molecule (guide RNA, gRNA). If we think of active Cas9 as a way to better understand genes (through deletions and mutations), then dCas9 is the route to get to know the genome a bit better – a particularly enticing mission for those, including myself, invested in the field of Genomics. From high-throughput targeted gene activation and repression screens to epigenome editing, dCas9 is helping scientists probe the genome in ways that weren’t possible before. Here, I put forth some of the best (in my humble opinion) applications, actual and potential, of CRISPR technology that go beyond genome editing.

 

Cas9 and Functional (Epi)Genomics

 

For many years the genome was considered as the totality of all genes in a cell; the additional junk DNA found was merely filler between the necessary gene units, stitching together chromosomes. We’ve come a long way since this naiveté, especially in recent years. We understand that the so-called junk DNA contains necessary regulatory information to get the timing and position of gene expression correct; and now, more than ever, we have a greater appreciation for the genome as a complex macromolecule in its own right, participating in gene regulation rather than acting as a passive reservoir of genetic material. The genome, it has been shown, is much more than just its sequence.

 

The epigenome, consisting of a slew of modifications to the DNA and the histones around which the DNA is wrapped, as well as the 3D organization of the genome in the nucleus, collaborates with DNA binding proteins to accurately interpret sequence information to form a healthy, functional cell. While mutations and/or deletions can be made – more easily, now, with Cas9 – to genomic sequences to test functionality, it is much harder to conduct comparable experiments on the epigenome, especially in a targeted manner. Because of the inability to easily perturb features of the epigenome and observe the consequences, our understanding of it is limited to correlative associations. Distinct histone modifications are associated with active versus inactive genes, for example; but, how these modifications affect or are affected by gene expression changes remains unknown.

 

Taking advantage of the tight binding properties of dCas9, researchers have begun to use the CRISPR protein as a platform to recruit a variety of functionalities to a genomic region of interest. Thus far, this logic has most commonly been employed to activate and/or repress gene expression through recruitment of dCas9 fused to known transcriptional activator or repressor proteins. Using this technique, scientists have conducted high-throughput screens to study the role of individual – or groups of – genes in specific cellular phenotypes by manipulating the endogenous gene locus. And, through a clever extension of the gRNA to include a hairpin bound by known RNA-binding proteins, the targeted functionality has been successfully transferred from dCas9 to the gRNA, allowing for simultaneous activation and repression of independent genes in the same cell with a single dCas9 master regulator – the beginnings of a simple, yet powerful, synthetic gene circuit.

 

Though powerful in its ability to decipher gene networks, dCas9-based activation and repression screens are still gene-centric; can this recruitment technique help us better understand the epigenome? The first attempts at addressing this question used dCas9 to target histone acetyltransferase, p300, to catalyze the acetylation of lysine 27 on histone 3 (H3K27) at specific loci. The presence of H3K27 at gene regulatory regions has been known to be strongly associated with active gene expression at the corresponding gene(s), but the direction of the histone modification-gene expression relationship remained in question. Here, Hilton et al. demonstrate that acetylation of regulatory regions distal to gene promoters strongly activates gene expression, demonstrating causality of the modification.

 

More recently, recruitment of a dCas9-KRAB repressor fusion to known regulatory regions catalyzed trimethylation of lysine 9 on histone 3 (H3K9) at the enhancer and associated promoters, effectively silencing enhancer activity. Though there have only been a few examples published, it will likely not be long until researchers employ this technique for the targeted analysis of additional epigenome modifiers. Already, targeted methylation, demethylation and genomic looping have been accomplished using the DNA-binders, Zinc Finger Nucleases and TALEs. With the increased simplicity in design of gRNAs, dCas9 is predicted to surpass these other proteins in its utility to link epigenome modifications with gene expression data.

 

Visualization of Genomic Loci

 

When you treat dCas9 as a bridge between DNA and an accessory protein, just as in the recruitment of activators, repressors and epigenome modifiers, there are few limits to what can be targeted to the genome. Drawing inspiration from the art of observation that serves as the foundation of scientific pursuit, researchers have begun to test whether dCas9 can be used to visualize genomic loci and observe their position, movements, and interactions simply by recruiting a fluorescent molecule to the locus of interest.

 

This idea, of course, is not entirely new. In situ hybridization techniques (ISH, and its fluorescent counterpart, FISH) have been successfully used to label locus position in fixed cells but cannot offer any information about the movement of chromosomes in living cells. Initial studies to conquer this much harder feat made use of long tracts of repetitive DNA sequence bound by its protein binding partner fused to fluorescing GFP; though surely an advance, this technique is limited because of the requirement to engineer the repetitive DNA motifs prior to imaging.

 

To circumvent this need, researchers have recently made use of TALEs and dCas9 (and here) carrying fluorescent tags to image unperturbed genomic loci in a variety of live cell cultures. The catch is that both TALEs and dCas9 perform much better when targeting repetitive regions, such that multiple copies of the fluorescent molecule are recruited, enhancing the intensity of the signal. Tiling of fluorescent dCas9 across a non-repetitive region using 30-70 neighboring gRNAs (a task made much more feasible with CRISPR versus TALEs) can similarly pinpoint targeted loci, albeit with much higher background. As is, the technique lacks the resolution desired for live imaging, but current advances in super-resolution microscopy and single-molecule tracking, as well as improvements in the brightness of fluorescent molecules available, will likely spur improvements in dCas9 imaging in the coming years.

 

Finally, dCas9 is not only useful in live cells. CASFISH, an updated Cas9-mediated FISH protocol, has been successfully used to label genomic loci in fixed cells and tissue. This updated version holds many benefits over traditional FISH including a streamlined protocol; but, most notably, CASFISH does not require the denaturation of genomic DNA, a necessary step for the hybridization of FISH probes, eliminating positional artifacts due to harsh treatment of the cells. Unfortunately, as of now, CASFISH also suffers from a need for repetitive sequences or tiling of gRNAs to increase signal intensity at the locus of interest.

 

Targeting RNA with Cas9

 

From cutting to tagging to modifying, it is clear that Cas9 has superstar potential when teamed up with double-stranded DNA (dsDNA); however, recent data suggests that this potential may not be limited to DNA. Mitchell O’Connell and colleagues at Berkeley found that Cas9 could bind and cleave single-stranded RNA (ssRNA) when annealed to a short DNA oligonucleotide containing the necessary NGG sequence. In addition, the authors made use of dCas9 and biotin-tagged gRNA to capture and immobilize targeted messenger RNA from cell extract. Though it remains to be shown, this proof-of-principle binding of dCas9 suggests that it is plausible to recruit a variety of functionalities to RNA as has been done for dsDNA. Recruitment of RNA processing factors through Cas9 could potentially enhance translation, generate known RNA editing events (deamination, e.g.), regulate alternative splicing events, or even allow visualization of RNA localization with conjugated fluorescent molecules. Again, each of these processes requires no modification to the RNA sequence or fixation, both of which can disrupt normal cell physiology.

 

Improving CRISPR Technology

 

The development of CRISPR technology, particularly the applications discussed here, is still in its infancy. It will likely take years of research for Cas9 and dCas9 to reach their full potential, but advances are underway. These developments pertain not only to the applications discussed here, but also genome engineering.

 

Specificity of Cas9

 

Cas9’s biggest flaw is its inability to stay focused. Off-target (OT) binding (and here) of Cas9 and DNA cutting have been reported and both present problems. With particular relevance to dCas9-based applications, promiscuous binding of Cas9 to regions of the genome that contain substantial mismatches to the gRNA sequence raises concerns of non-specific activity of the targeted functionality. Efforts to reduce OT binding are needed to alleviate these concerns, but progress has been made with the finding that truncated gRNA sequences are less tolerant of mismatches, reducing off-target Cas9 activity, if not also binding.

 

Temporal Precision of Cas9

 

One of the most exciting developments in dCas9 genome targeting is the potential to manipulate the genome and epigenome in select cell populations within a whole animal to gain spatial resolution in our understanding of genome regulation; however, as we have learned over the years, gene expression patterns don’t only change with space, but also time. A single cell, for example, will alter its transcriptome at different points during development or in response to external stimulus. The development of split versions of Cas9 (and dCas9), which require two-halves of the protein to be expressed simultaneously for function, will not only improve spatial specificity of Cas9 activity but holds the potential to restrict its activity temporally. Drug-inducible and photoactivatable (!) versions of split Cas9 restrict function to time windows of drug treatment or light activation, respectively. In addition, a ligand-sensitive intein has been shown to temporally control Cas9 activity by releasing functional Cas9 through protein splicing only in the presence of ligand.

 

Expanding the CRISPR Protein Repertoire

 

Finally, CRISPR technology will likely benefit from taking all of the weight off of the shoulders of Cas9. Progress toward designing Cas9 molecules with altered PAM specificity, as well as the isolation of Cas9 from different species of bacteria, has helped expand the collection of genomic sites that can be targeted. It has also enabled multiplexing of orthogonal CRISPR proteins in a single cell to effect multiple functions simultaneously. More recently, the Zhang lab isolated an alternative type II CRISPR protein, Cpf1, purified from Francisella novicida. Cas9’s new BFF is also able to cut genomic DNA (as shown in human cells), but in a slightly different fashion than Cas9, generating sticky overhangs rather than blunt ends. Cpf1 also naturally harbors an alternate PAM specificity; rather than targeting sequences upstream of NGG, it prefers T-rich signatures (TTN), further expanding the genomes and genomic sites that can be targeted.

 

CRISPR/Cas9 has already proven to be one of the most versatile tools in the biologist’s toolbox to manipulate the genomes of a variety of species, but its utility continues to grow beyond these applications. Targeting Cas9 to the mitochondria rather than the nucleus can specifically edit the mitochondrial genome, with implications for disease treatment. Cas9 has been used for in vitro cloning experiments when traditional restriction enzymes just won’t do. And, by directly borrowing the concept of Cas9 immunity from bacteria, researchers have enabled enhanced resistance to viruses in plants engineered with Cas9 and gRNAs. While we ponder what innovative technique will come next, it’s important to think about how this cutting-edge technology that promises to bolster both basic and clinical research came to be: this particular avenue of research was paved entirely by machinery provided by the not-so-lowly bacteria. That’s pretty amazing, if you ask me.