CRISPR Digest

Since I last wrote about the current CRISPR Craze, the new editions of Nature, Science and Cell have all featured new CRISPR innovations and advances, which I will outline here in an attempt to give a glimpse into the latest research in the genome-engineering field.

For a good general introduction, including both the discovery and recent applications of the CRISPR/Cas9 system, I recommend the review by Hsu et al. (2014) published early this summer.

One of the applications discussed in this review is the so-called CRISPR interference (CRISPRi, in the style of RNA interference/RNAi) method. It was first developed by Qi et al. (2013) and relies on a catalytically inactive Cas9 nuclease (termed dCas9). dCas9 is targeted to a gene of interest using a guide RNA (gRNA), just like in the original mechanism, except that here the dCas9 cannot cleave the double-stranded DNA, but instead physically blocks the RNA polymerase from transcribing the gene, thus reducing/abolishing the expression of the target gene. One of the advantages of this method is that dCas9 expression is inducible, enabling the system to act as an on/off switch for the gene of interest. Furthermore, dCas9 can also be fused to transcriptional activators and this serves to turn on/overexpress genes of interest.

Due to the high efficiency and specificity of the system [beware, however, that of course the system also has off-target effects; see, for example, Lin et al. (2014)]  it has also been utilised for genome-scale CRISPR/Cas9 knockout (GeCKO) screening experiments. In the experiments described by Shalem et al. (2014), CRISPR is used to knock out almost every single gene in the human genome individually. They did this in a melanoma cell line, which harbours the BRAF V600E mutation, and which is therefore sensitive to the BRAF kinase inhibitor vemurafenib: after treatment with the drug most cells died as expected, but those that survived (i.e. had become resistant to vemurafenib) were analysed. Among the survivors  they found gRNAs targeting genes whose loss had previously been implicated in resistance, but they also found genes that had not been linked to resistance yet. Genome-wide screens like this can thus provide the starting points for more detailed, mechanistic studies into various biological processes, including drug resistance mechanisms.

Even more recently Feng Zhang’s group at MIT reported (Platt et al. (2014)) an engineered mouse line expressing the Cas9 nuclease specifically in different tissues (again beware of “specific”: tissue-specific promoters are rarely exclusive to a single tissue type). These mice were used for ex vivo manipulation of dendritic cells (cells of the immune system): after bone marrow cells were harvested, the dendritic cells (which already express Cas9) were infected with a (lenti)virus carrying the gRNA and such edited cells could then theoretically be reintroduced into the mice. They also showed that in vivo experiments can be performed with these mice when the virus expressing the gRNA is directly injected into the brain, for example, or used to make multiple changes in the genomes of lung cells to model cancer initiation/progression.

And only yesterday a paper was published in Nature by O’Connell et al. (2014), showing that CRISPR can also be used for RNA recognition and cleavage, as opposed to DNA cleavage. This can be achieved when the so-called protospacer adjacent motif (PAM), which is required for the recruitment and activation of  the Cas9 nuclease, is provided alongside the gRNA in the form of a short DNA sequence. It may now become feasible to use CRISPR to complement RNAi approaches. The possibilities are endless.

References:

Hsu PD, Lander ES, Zhang F (2014) Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell 157: 1262-1278

Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research 42: 7473-7485

O’Connell MR, Oakes BL, Sternberg SH, East-Seletsky A, Kaplan M, Doudna JA (2014) Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature advance online publication

Platt Randall J, Chen S, Zhou Y, Yim Michael J, Swiech L, Kempton Hannah R, Dahlman James E, Parnas O, Eisenhaure Thomas M, Jovanovic M, Graham Daniel B, Jhunjhunwala S, Heidenreich M, Xavier Ramnik J, Langer R, Anderson Daniel G, Hacohen N, Regev A, Feng G, Sharp Phillip A, Zhang F (2014) CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Cell – in press

Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell 152: 1173-1183

Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science 343: 84-87

#OA = Open Access

 

Open-Access-logo

This is the symbol for open access (image from PLoS, the Public Library of Science). Two days ago Nature Communications, a “journal that publishes high-quality research from all areas of the natural sciences” within the larger and prestigious Nature Publishing Group, announced that it will only be accepting open access (OA) submissions starting next month. Yesterday the Royal Society in London tweeted to announce the launch of their new OA journal Royal Society Open Science.

So what exactly is OA publishing? Simply put, it is the online publishing of research results that does not put any restrictions on who can read the articles, and additionally, allows further distribution and re-use of the material as long as the original source is cited. This relatively new model of publishing stands in contrast to the well-established traditional publishing model, in which readers can only access journal contents when they (or the institutions they work for) pay subscription fees. Generally, there are two types of OA publishing (for more information and resources I found the site of Peter Suber very helpful):

  1. “gold OA”: in the form of OA journals in which the papers are peer-reviewed before publication; examples include PLoS and BioMed Central (for a full list see the Directory of OA Journals)
  2. “green OA”: in the form of OA repositories in which articles are stored online and may or may not have gone through a peer-review process

If the reader no longer pays for access to information, who provides the money for the editing and publishing process? There are several models to finance OA publishing. A common model has the authors (or the authors’ institutions) pay for their publication once the paper has been accepted; this usually occurs in the form of an Article Processing Charge (APC). These fees are far from negligible: Royal Society Open Science charges £1000, PLoS Biology approximately £1800 and Nature Communications a whopping £3150.

The first drawback of OA immediately becomes obvious: not all researchers have a budget large enough to pay for the publication of their work after having spent a lot of money on the research itself. Does this mean that OA is biased towards making big and successful labs even more successful? Thankfully, OA journals usually offer to waiver the APC if the research group is based in a low-income country, and often large funding bodies support their scientists by paying for the APC as well. A way to think of the concept is to liken it to social security: those that can pay for their publications, and in doing so also support those that cannot afford the APC as well as providing money for the review process of those manuscripts that are eventually rejected.

Another concern that arises is so-called “predatory OA”, in which publishers try to make a profit from the APC without rigorously reviewing and editing papers, thus increasing the amount of badly curated information available on the internet. To counteract this trend Jeffrey Beall regularly updates a list of journals that are “questionable”.

Lastly, the question arises of whether more people actually read journal articles just because they can. Do laypeople read about discoveries on diseases in PLoS Pathogens or about replication studies and negative results in PLoS One? I don’t know the answer to this, but probably not. OA is partly an ideological step forward: who pays for most of the research conducted in institutions and universities? Taxpayers. So who should be allowed to read the outcome of this research? Taxpayers.

Assuming that laypeople do not benefit from OA, who does? Scientists/academics at small institutions that cannot afford to buy subscriptions to all journals; recent graduates that want to keep up with current scientific literature but no longer have access via their institutions; doctors with private practices that are not affiliated with hospitals: only a few days ago my mother tried to find information on the treatment of a complicated case of endometriosis (a disease in which cells of the uterine lining grow outside the uterine cavity), but she couldn’t access papers published in The Lancet or The New England Journal of Medicine, so I had to help her out by signing in using the university’s subscription.

Another perk of online OA publishing is that it is less restrictive with regards to the length of the papers published. Unlike in journals such as Science, where high impact research is squeezed on to three pages (with dozens of pages of supplementary material), OA journals can afford to “print” long articles enabling scientists to show off all their hard work.

What will the future trend be? Will all journals become OA or will there continue to be subscription-only publishing in science? From what I have read and experienced there is certainly a tendency towards more OA, also among the established high impact factor journals in the form of green OA (making the paper available six months after initial publication). Additionally, there are archives that make papers available before they are printed (e.g. arXiv for physics and computational sciences or bioRxiv for biology). It remains to be seen what long-term benefits OA publishing will have.

From Bench to Bedside…

… is an evocative phrase that almost has one believe that busy scientists in their immaculate white lab coats, bustling about a clean high-tech lab filled with glass flasks containing brilliantly blue and luminescent yellow solutions, are delivering “magic bullet” drugs directly to their colleagues in the clinic. In my, admittedly limited, experience nothing could be further from the truth. When I tell people that I “did cancer research” this summer, investigating the differences between primary and metastatic prostate cancer (PC) in a lab at Cold Spring Harbor Laboratory, it sounds a lot more glamorous than it is.

There are probably two major factors that contribute to this somewhat idealised notion of how translational science works. Firstly, I would wager that the majority of the general public is not aware of what a long-winded process scientific discovery is. In the cancer research field, for example, basic cellular processes are usually probed and tested in cell culture models to begin with. If these experiments yield interesting results then the next step will involve using an animal model (often mice) to test whether the previously formed hypotheses hold true in vivo. If, either by sheer luck or rational thought, a drug is identified that can interfere with the process under scrutiny then it can be tested pre-clinically. If this is successful – and the definition of “successful” in this case is complex because of the guidelines from the Food & Drug Administration, for example – then maybe the drug can be used in Phase I (out of III) clinical trials. The clinical trials are expensive and can take several years to carry out and evaluate. So in total it usually takes at least a decade (and this is most likely a very optimistic estimate) for some basic discovery to gain clinical relevance. Of course there are exceptions to this, and despite the length of the process it is still certainly worth doing.

Secondly, I think that a lot of researchers investigating some disease or other are often unaware of the clinical/medical/social/personal implications that a disease has. A few days ago a close friend of my mother’s, who was diagnosed with PC earlier this year, asked me whether I could tell him more about the research I had done and more about PC in general. I didn’t know what to say. Presumably he didn’t really want to hear about the molecular pathways (e.g. PTEN/Akt pathway) involved. Nor is it particularly interesting to an Austrian individual what the current incidence and mortality rate statistics are in the USA. Reading about novel therapeutic agents that are just starting to be tested and won’t be ready for widespread use anytime soon isn’t going to be helpful either. I ended up sending him some review papers that focus on the clinical aspects of PC (e.g. Heidenreich et al. (2013)), but essentially I was helpless. Working with and experimenting on a cell line that, once upon a time, came from a bone metastasis of a patient with PC just isn’t the same as being confronted with a whole human being suffering from the disease.

In summary, therefore, I think more communication between both sides, and education on both sides, are needed. Patients lying on the bed or people standing by the bedside (doctors and relatives alike) often don’t know about how research at the bench works, and understandably, knowledge about basic research isn’t necessarily a priority for them at such a difficult time. Conversely, people standing at the bench usually aren’t doctors and don’t know about the disease, except in a defined experimental setting. Even MD/PhD researchers need to go through the laborious process outlined above.

Are there any ways of improving the situation? As a layperson: ask more questions. As a scientist: learn better ways of communicating, and maybe to enrol in a “Bench to Bedside” PhD programme such as the one offered by UCL.

Reference:

Heidenreich A, Pfister D, Merseburger A, Bartsch G (2013) Castration-resistant prostate cancer: where we stand in 2013 and what urologists should know. European urology 64: 260-265

Non-Academic Careers for Scientists

Now that you’ve finally decided to definitely pursue a PhD in your chosen field you’re incredibly excited to get started. You’ve created a new EndNote library devoted to your graduate studies and bought new stationary, enabling a fresh and energetic start. But wait, what’s this? A friend of yours shares this explanation as to what a PhD actually is (animated by Pavel Boytchev, from The Illustrated Guide to a PhD by Matt Might):

Suddenly you feel humbled, and at the same time even more excited to start pushing that boundary just a tiny little bit further. But then, creeping up, there is also  the feeling of slight disillusionment because you always enjoyed learning about lots of different things and you know that that’s not what a PhD will offer. You’re afraid that you’ll lose sight of the big picture, so maybe after doing a PhD (and possibly even a post-doc) you want to zoom out from that pinprick of knowledge. How exactly are you proposing to do that? Well, I don’t know myself, but after having read a preview of this book (image from CSHL Press website) I am intrigued by the editing/science writing/publishing field. Who knows, maybe there’s also a chapter that will catch your fancy?

CareerOpBioSci_fNow I am certainly getting ahead of myself though; PhD applications, here I come!

 

 

Max Perutz Interviews

The Vega Science Trust – Max Perutz Interview 1 – watch the first 45 seconds to hear about Perutz’s motto, “in science, truth always wins”. And then maybe watch the rest, indulging yourself in productive procrastination.

The Vega Science Trust – Max Perutz Interview 2 – here Max Perutz talks about science (with an emphasis on crystallography of course) and other important topics, such as religious fundamentalism (9:30) and faith/atheism (42:40) and the problem of overpopulation, partly due to lack of birth control. I particularly agree with him that, as a scientist, one should take a stance against creationism, but also not go as far as people like Richard Dawkins who relentlessly attack religion, which, Perutz says, “discredits science” and “shows our arrogance”.

Also watch out for an instance of mouth pipetting at 4:45 in the video posted in the last entry (https://lovecells.wordpress.com/2014/09/14/in-science-truth-always-wins/) – this would be unheard of today!

Lastly, Perutz made an interesting prediction about whether and when the protein structure of haemoglobin would be solved in a paper from 1949:

Untitled

[from: Perutz MF (1949): Recent developments in the x-ray study of haemoglobinRoughton FJWKendrew JC, eds. Haemoglobin: A Symposium Based on a Conference Held at Cambridge in June 1948 in Memory of Sir Joseph BarcroftLondonButterworths Scientific Publications135147]

Funnily enough, the structure of haemoglobin was eventually solved in 1968 – a year before the Apollo 11 moon landing.