CRISPR Digest #5

Edit on April 23rd 2015: Just three days after posting about “a prudent path forward for genomic engineering” I read this Nature News & Comment article describing the experiments of a research group in Guangzhou, China in which they used CRISPR/Cas9 technology on human embryos (Liang et al. (2015)). [Their article was published open access in Protein Cell and can be found here.] Admittedly, the embryos they used came from so-called tripronuclear zygotes, which are zygotes formed when an egg cell is fertilised by two sperm cells as can happen during in vitro fertilisation. These embryos are therefore not viable in vivo. However, the question remains whether this is “prudent” use of CRISPR technology. On the one hand, yes, if genome-editing in humans is ever going to happen on a regular basis then we need to understand exactly how it works. For instance, they do show that their approach has considerable off-target effects: their guide RNAs were targeting the beta-globin gene (which encodes a subunit of haemoglobin) but DNA breaks were also induced at other sites. On the other hand, experimenting on human embryos will (probably) always be scrutinised with ethical concerns in mind, especially because it is really not clear that genome-editing in human embryos should be the way forward.


Since the last CRISPR update there have been some developments regarding this new genome-editing technique. Leading scientists in the field (Baltimore et al (2015)) met in Napa, California at a bioethics conference organised by the Innovative Genomics Initiative (IGI) to discuss CRISPR policy and make discussion of this topic more visible to and inclusive of doctors, social scientists and the public. They pinpoint four recommendations to be put into immediate action:

  1. Strong discouragement of “any attempts at germline genome modification for clinical application in humans, while societal, environmental, and ethical implications of such activity are discussed among scientific and governmental organizations”.
  2. Creation of forums of experts in science and ethics to discuss the potentials and risks of this technology.
  3. Transparent research to gain a better understanding of how CRISPR works.
  4. Creation of a “globally representative group of developers and users of genome engineering technology […] to recommend policies”.

I noticed that Feng Zhang of MIT was not a co-author of this paper (and neither was Emmanuelle Charpentier, to my surprise), but maybe this is because he is now or will soon be involved in a “winner-take-all” patent dispute. At the moment, Zhang (and MIT/Broad Institute?) own the rights to CRISPR and claim that they invented the technology first/were the first to make it work. However, their patent application was filed half a year after the Doudna/Charpentier patent in 2012. So now the University of California in Berkeley (where Doudna works) and the University of Vienna (where Charpentier worked) set up a so-called patent interference request and if they are successful they will own CRISPR rights entirely, leaving Zhang with nothing. The whole process is summarised here, from where I also copied this figure, showing the number of papers published on CRISPR in the past ten years:

crispr paper numbers

The graph illustrates how much is at stake during this patent interference process. Equally, the technology is getting attention in non-scientific circles: Time Magazine’s 100 most influential people include Jennifer Doudna (left) and Emmanuelle Charpentier (right) in the “pioneer” category. Here they are last year at the Breakthrough Prize in Life Sciences ceremony probably looking very much the opposite of what society at large thinks scientists (should) look like:

Life Sciences co-laureates Doudna and Charpentier speak on stage during the 2nd Annual Breakthrough Prize Awards in Mountain View

References:

Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M, Greely HT, Jinek M, Martin GS, Penhoet E, Puck J, Sternberg SH, Weissman JS, Yamamoto KR (2015) Biotechnology. A prudent path forward for genomic engineering and germline gene modification. Science 348: 36-38

Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J (2015) CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell: 1-10

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The Joys of Revision #1

Please immediately note the hint of sarcasm in this title. What academic year would be complete without the harrowing prospect of end-of-year exams, which, this year, happen to also be finals? … Well, exactly. Any old academic year would be fine without. But alas, that is not how the system works. So on top of being judged on the basis of our lab project/dissertation/viva voce examination we also need to take two written exams, which are worth 50% of our mark.

To ease the transition from dissertation writing and editing into revision I thought I would start with something fun/potentially useless that can be classed as elaborate procrastination:

Landmark timeline

As mentioned previously, some of our teaching was provided in the format of seminars discussing landmark papers – papers that have shaped the way biologists think about their areas of research and that have had lasting, even if sometimes subconscious, effects. Several of the professors emphasised the importance of understanding these papers within their historical contexts, which is, I would contest, not something we scientists normally think about. Therefore I decided to look for a tool that would allow easy creation of a timeline like the one shown above and the only free app I could find in the store is simply called Timeline 3D. Right, well, and I just realised that on top of procrastinating by colour-coding different landmark papers I’ve now also spent a good fifteen minutes writing this blog post. If anyone else has well-disguised procrastination tools, please do share…

P.S.: Another way to pretend like you’re doing something useful – attend the departmental seminar given by a Nobel prize laureate, Jules Hoffmann. I was pleased to hear that he was appreciative of all of his collaborators and acknowledged his students and post-docs; something he seems to share with another Nobel prize winner, Christiane Nüsslein-Volhard, who gave a seminar a few weeks ago. Based on these examples I was about to draw the conclusion that a couple of the marks of great scientists are a) their willingness to acknowledge co-workers and b) to some extent their modesty (granted, this one doesn’t apply to other Nobel laureates I’ve encountered [e.g. James Watson]). However, as I was reading about Jules Hoffmann I very quickly came across this blog written by Bruno Lemaitre, a former research associate in Hoffmann’s lab, claiming that most, if not all, the award-winning work was conducted by him without encouragement/help from Hoffmann. Now what to believe?!

Cancer Discovery

As of March 31st 2015 I am the proud co-author of “Myc drives Pten/p53-deficient proliferation and metastasis due to Il6-secretion and Akt-suppression via Phlpp2” – snappy title, right? – which was published “online first” in the journal Cancer Discovery. The abstract and author manuscript can be found here. The polished article will appear in the May print issue, I believe.

The first author, Dawid G. Nowak, was my supervisor at Cold Spring Harbor Laboratory this summer, where I participated in a ten-week undergraduate research programme. The main aim of the paper was to characterise molecular differences between prostate cancer (PC) cells of the primary tumour (i.e. at the prostate) and metastasised prostate cancer cells. A well-known change that occurs during the progression of PC is the loss of so-called tumour suppressor genes, in particular they are the proteins called PTEN and p53. Tumour suppressor genes are responsible for preventing healthy cells from overproliferating, so when they are lost there are fewer “molecular brakes” in the cell to stop aggressive growth. In addition to losing tumour suppressors, cancer cells also activate so-called oncogenes, and these can be viewed as drivers of growth. So, broadly speaking, when tumour suppressors are lost and oncogenes constitutively switched on a cancer cell is created.

Dawid’s work shows for the first time that as PC cells become metastatic they switch from using one main “driver” oncogene known as Akt to using a different oncogene known as Myc. Firstly, they just made this observation in various tissue samples and biopsies, but then the lab, led by Lloyd Trotman, tried to elucidate the mechanism by which this “oncogene switch” occurs. What Dawid found was that an extracellular signalling factor called IL-6, normally involved in inflammatory processes, activates Myc, which in turn activates a phosphatase – an enzyme that dephosphorylates its targets – called Phlpp2. One of Phlpp2’s targets is Akt, and Akt is inactive when dephosphorylated. Thus, once this process is set into motion Myc rapidly overtakes Akt as the main driving oncogene.

Needless to say, one of the methods employed in the study was the CRISPR knockout approach. The idea of the CRISPR experiment was to show that in a PC cell line derived from a patient’s bone metastasis (PC3), which has already lost the tumour suppressors PTEN and p53, we could knock out Myc and at the same time decrease the levels of Phlpp2. And indeed this is what the following Western blot shows (copied directly from Figure 6 of the paper) – on the left is the control and on the right the CRISPR approach has completely knocked out Myc; PCNA is a marker for cell proliferation and beta-actin is the loading control, which shows that equivalent amounts of total protein are present in both lanes:

Screen Shot 2015-04-06 at 21.26.55

And then in the obverse experiment Myc was overexpressed in these PC3 cells (again the Western blot is copied from Figure 6 and the left is the control, whereas the right is the experiment). When this was achieved the levels of Phlpp2 also increased and furthermore, the levels of phosphorylated/active Akt decreased:

Screen Shot 2015-04-06 at 21.27.01

One could now argue, however, that this was merely a correlation but not a causative effect. So to prove that the proposed signalling – Myc activates Phlpp2, which inactivates Akt – was actually occurring, the last experiment was performed in PC3 cells in which, in addition to PTEN and p53, Phlpp2 had also been deleted. Here the levels of phosphorylated Akt no longer decreased because the responsible phosphatase was not present (again from Figure 6; left: control, right: Myc overexpression):

Screen Shot 2015-04-06 at 21.33.00

All in all quite an elegant story I think. Hopefully the scientific community studying PC will be able to use these results in further experiments. For my part I certainly learnt a great deal from Dawid last summer and enjoyed helping to prepare the manuscript for publication.

Reference:

Nowak DG, Cho H, Herzka T, Watrud K, DeMarco DV, Wang VM, Senturk S, Fellmann C, Ding D, Beinortas T, Kleinman D, Chen M, Sordella R, Wilkinson JE, Castillo-Martin M, Cordon-Cardo C, Robinson BD, Trotman LC (2015) MYC Drives Pten/Trp53-Deficient Proliferation and Metastasis due to IL6 Secretion and AKT Suppression via PHLPP2. Cancer Discovery 5: 636-651

A Science Masterclass

For the past three days I have been acting as a so-called student ambassador during an Easter residential science masterclass. This was a programme aimed at 14- to 16-year old students (about to take their GCSE exams) from across several areas of England, including Bedfordshire, Leicestershire, Northamptonshire and Southwark (London); the students stayed in Pembroke College accommodation for two nights and participated in a range of scientific activities. The students from these “link areas” were nominated by their teachers and attend schools that may not have particularly strong connections with any universities, and Oxbridge in particular. The idea of these Access and outreach programmes is to encourage students from less privileged backgrounds to consider applying to university, including Cambridge. In part this is achieved by showing them that Cambridge is not only for posh, rich, upper-class students (although they do also exist).

We started the masterclass with a tour of Pembroke College – did you know that the college was founded in 1347, but did not admit women as members until 1983? Or that one of our most famous graduates, William Pitt the Younger, became Prime Minister at the age of 24? Or that there are 116 libraries scattered around Cambridge?

Next, there was a treasure trail through the historic city centre – although “treasure” might have been a misnomer depending on your inclinations, since each station consisted of working through a maths problem. We saw Newton’s apple tree outside Trinity College and The Eagle pub, where Francis Crick and James Watson allegedly had the insight into “the secret of life”, the structure of DNA. [We discussed the contribution of Rosalind Franklin, of course!] Dotted in between all these activities were various brainteasers, such as trying to solve the Monty Hall problem or Einstein’s puzzle (have a go – they are surprisingly fun).

In addition to the activities organised by the college’s school liaison officer there were outings to various University departments: we examined the relationship between leaf surface area and volume in both desert and tropical plants at the Botanic Gardens; we compared femur length and diameter in dinosaurs of varying sizes at the Sedgwick Museum of Earth Sciences and came to the conclusion that there is a natural limit to the size of land animals, since at some point their femurs would have to become wider than they are long to support their weight. And as if that was not already enough science for a day we walked to the Cavendish laboratory, where I attended my first ever physics lecture:

image1

The day was rounded off by a talk on higher education, a formal dinner (juice instead of wine) and an evening with Sir Isaac Newton. However, the most striking point was really that the English secondary school education system can be extremely restrictive: if you want to apply for a science degree at university then essentially you have to take only science A levels; a girl asked about whether she could combine science and drama, but essentially she was advised against it and that was a slightly heartbreaking moment. On the other hand, choosing only science subjects means you can focus more and maybe gain a deeper understanding of those few subjects, but should that really happen at the expense of other interests? And at such an early age?

In any case, our last big activity took us to the plant sciences department where we extracted chloroplasts from lettuce leaves, measured their sizes and investigated whether a certain herbicide can inhibit photosynthesis – it was fun to teach them how to use a Gilson pipette (known as an Eppendorf pipette in German-speaking Europe) despite being slightly shocked by the fact that some people do not already know how to use them…

image2

Overall, the students on the programme were really a pleasure to supervise – asked interesting questions, engaged with my questions, had a good sense of humour – and made my first teaching experience both exhausting (in a good way) and enjoyable.