About Victoria Wang

A PhD student at The Francis Crick Institute in London; avid reader; occasional writer in the hope that writing will help both me and others understand the world better. If you're interested check out my twitter @vwang93

CRISPR Digest #14

Two years ago, in spring 2015, Liang et al. published the first report of gene-editing in human embryos using CRISPR/Cas9 (mentioned previously here, here and here). At the time no high-profile journal was willing to take on the risk of publishing what was perceived to be a controversial study. Liang et al. were trying to correct mutations in the human beta-globin gene – mutations in this gene can lead to a group of diseases called beta thalassaemias, including sickle cell anaemia – in human embryos that had been fertilised by two sperm cells (and could therefore never develop). In fact, the take-home message from their study was that using the techniques available to them at the time led to a host of unwanted side effects, including the creation of mutations at other sites in the embryo genome and the “correction” of the beta-globin gene with a similar gene called delta-globin.

Last month, a different group (Ma et al. – four first authors and five corresponding authors!) published more work on human embryo CRISPR/Cas9 gene-editing, this time in Nature. Like Liang et al. this paper also tried to tackle a monogenic disease, a disease that is caused by a well-defined mutation in a single gene, called hypertrophic cardiomyopathy. The affected gene is MYBPC3 and when mutated (denoted as DeltaGAGT in the figure below) this leads to a thickening of the heart muscle, which in turn can cause heart failure. The authors used donor sperm with the MYBPC3 mutation together with healthy oocytes to perform their experiments. In the first approach the eggs were fertilised by the sperm and only subsequently, during S phase, were the guide RNA, Cas9 protein and a piece of non-mutated donor DNA injected. The guide RNA was designed to specifically recognise the mutant version of MYBPC3, which recruits the Cas9 protein to make a cut in the DNA, and then the donor DNA would serve as a template to repair the sperm’s mutated gene. Ma et al. observed that this technique worked but often generated so-called mosaic embryos, which contained a mixture of healthy and mutated cells. This incomplete gene correction happened because during S phase both the maternal and paternal chromosomes duplicate and therefore the CRISPR/Cas9 system would have to correct two mutated MYBPC3 genes before the first cell division.

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Schematic depicting CRISPR/Cas9 stage at zygote stage (top) versus together with sperm (bottom) – copied directly from Ma et al, 2017

In a second approach, Ma et al. wanted to overcome this mosaicism by injecting the CRISPR components together with the sperm during the M phase of the oocyte. Now only one copy of mutant MYBPC3 had to be corrected and this succeeded in producing completely healthy embryos. Ma et al. also checked to make sure that these embryos did not carry any unwanted, off-target mutations.

Last but not least, Ma et al. provided evidence that often the human zygote used the healthy maternal gene to provide a template for the repair of the mutated paternal gene, instead of the injected DNA template. This is significant because in most cell types the DNA double-strand breaks caused by Cas9 are usually repaired in an imprecise manner (called non-homologous end joining) and lead to further mutations. Ma et al. therefore argued that “human gametes and embryos employ a different DNA damage response system”.

This finding could be of huge importance, both to the basic understanding of human embryonic development as well as to potential therapeutic CRISPR/Cas9 applications. However, four days after the Nature paper was published online, several prominent scientists posted a riposte on the pre-print server bioRxiv. Egli et al. criticised the first paper quite heavily by raising theoretical objections/concerns; they couldn’t have tried to replicate the experiments in such a short time frame. [Note that this pre-print was, of course, not peer-reviewed, although the authors have confirmed that they were trying to get their work published in Nature as well.]

Among other more technical issues to do with the way in which healthy and mutant genes were detected, Egli et al. pointed out that after fertilisation the maternal and paternal chromosomes remain physically separated (indicated by the arrows in the figure below) until just before the first cell division. Therefore, Egli et al. argued, it is highly unlikely that the healthy maternal MYBPC3 gene could serve as a template for the repair of the mutant paternal gene. This strikes me as a strong argument, not being at all familiar with early human development. Overall, Egli et al. suggested that Ma et al. were simply not detecting the mutant gene in their embryos but not providing good enough evidence of a corrected gene. The scientific debate will, no doubt, continue and I think having bioRxiv as such a rapid place for the exchange of ideas can drive scientific discourse.

Egli et al - early development
Pictures of a human zygote (fertilised egg/oocyte) and its very early development – copied directly from Egli et al, 2017

Since this is a digest it should also contain some other relevant CRISPR/Cas9-related news. One of the post docs I met at Cold Spring Harbor Laboratory in 2014, Serif Senturk, published a paper early this year in which the authors show how they can switch CRISPR on or off in living cells. They did this by fusing the Cas9 protein to another, destabilising protein domain, which caused the attached Cas9 to get degraded. However, when a “shield molecule” was added to the cells, the destabilising domain was no longer active and the Cas9 could accumulate. This innovation counteracts the problem of off-target effects, which are often due to the long duration that Cas9 is active for. Pretty neat system, I think.

Senturk 2017

Schematic depicting Cas9 fused to a destabilising domain – copied directly from Senturk et al, 2017


References:

Egli D, Zuccaro M, Kosicki M, Church G, Bradley A, Jasin M (2017) Inter-homologue repair in fertilized human eggs?
bioRxiv: http://www.biorxiv.org/content/early/2017/08/28/181255

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

Ma H, Marti-Gutierrez N, Park SW, Wu J, Lee Y, Suzuki K, Koski A, Ji D, Hayama T, Ahmed R, Darby H, Van Dyken C, Li Y, Kang E, Park AR, Kim D, Kim ST, Gong J, Gu Y, Xu X et al. (2017) Correction of a pathogenic gene mutation in human embryos. Nature 548: 413-419

Senturk S, Shirole NH, Nowak DG, Corbo V, Pal D, Vaughan A, Tuveson DA, Trotman LC, Kinney JB, Sordella R (2017) Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization. Nature Communications 8: 14370

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PhD – 21 months in 

Do you remember my optimistic blog post about finding my bearings in the lab after a month of the PhD? I also included pictures of a failed western blot and slightly crushed centrifuge tubes.

Well, twenty months later and I’m still making mistakes. Often they’re new and different mistakes, which could almost be exciting. But today I made the same mistake and lost a lot of plasmid-growing bacteria (bacteria I am using as work horses to produce specific DNA for me) in a centrifuge (which I subsequently cleaned!)…

Photographic evidence attached.

11th International PhD Student Cancer Conference

A glorious three day bonanza of beer, brains and BRAF. — Tom Mortimer, PhD student at The Francis Crick Institute

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On Wednesday morning, June 14th, twenty PhD students from The Francis Crick Institute woke up early and made their way from one of London’s five airports to Berlin. Specifically to Campus Berlin-Buch – the geographic equivalent of Clare Hall Laboratories, situated right next to the M25, the London Orbital Motorway, 25 kilometres from the city centre – home to the Max Delbrück Center for Molecular Medicine (MDC).

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On the campus of the MDC

We were attending the 11th international PhD student cancer conference (IPSCC), which was initiated at the London Research Institute (LRI), one of the founding partners of The Crick. In fact, the opening remarks were held by Holger Gerhardt, a former group leader at the LRI. He immediately gave the meeting a political flavour by stressing how important diversity is within research, openly showing his disdain for Brexit.

The conference was organised by PhD students at the MDC for other students studying cancer across Europe, with delegates from the UK, Germany, Italy and the Netherlands. The talks were spread over three days and the topics ranged from in silico computational biology and large-scale genomics approaches to cell signalling and in vivo cancer metabolism. Strikingly, when speakers were given suggestions or asked questions they seemed sincere in their responses, especially when they didn’t know the answers. One of the talks most out of the ordinary was given by Joseph Hodgson from the CRUK Beatson Institute in Glasgow: he uses fruit flies to study the process of weight loss and muscle wasting due to cancer (also known as cachexia).

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Joseph Hodgson showing fluorescent images of fruit fly muscle wasting (right)

The prize for the best talk went to Rajbir Nath Batra, from the CRUK Cambridge Institute, who studies DNA methylation dynamics in breast cancer in Carlos Caldas’ group. The best poster by far was created by Cora Olpe, also at the Cambridge Institute, who is trying to understand the chemopreventive effect of aspirin on colorectal cancer in the group of Douglas Winton.

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Cora Olpe’s poster made use of Aspirin’s chemical formula to great effect

On the social side of things, conversation was enabled by providing generous amounts of delicious German beer as well as having us participate in career workshops, including on grant writing, conducting clinical trials, science communication and on becoming an entrepreneur. All in all it was great to get the opportunity of meeting the people who might be our future collaborators.

The keynote speakers were Mónica Bettencourt-Dias (Gulbenkian Institute, Lisbon) and Madalena Tarsounas (Institute for Radiation Oncology, Oxford). Lastly, Klaus Rajewsky (MDC, Berlin), a world-renowned immunologist, gave a lecture on his “life in science”. He ended the conference also on a political note, juxtaposing the 1975 referendum on the UK’s membership to the European common market with the Brexit referendum, also stressing how important international collaboration and diversity are within science.

Next year the 12th IPSCC will be hosted by The Francis Crick Institute. We hope to have a great turnout (especially in the face of Brexit) – see you there!

 

March for Science

London, Saturday April 22nd 2017

The weather is changeable as I leave the flat in the late morning. Sunny spells – dazzling my eyes clad in contact lenses – are abruptly overtaken by the English drizzle that leaves me damp and puzzled because the sun has already regained its prominence. I’m on the Westbound Piccadilly line wearing a Cancer Research UK t-shirt that reads, “I’m a researcher fighting cancer”, and I can’t tell whether I’m getting more looks than is usual on the Tube. I alight at South Kensington to meet a friend of mine, the bubbleologist Li Shen. (And yes, that is now a technical term. Li, who has a degree in mathematics, is a PhD student studying the physics of bubbles, which has far-reaching implications: from the amount of bubbles generated by different types of beer to the undesired foaming of lubricants used in oil extraction.) But we’re not just here to catch up, although it is conveniently close to his lab/office at Imperial College. No, we’re here to join the March for Science. [All of the following images were taken either by Li or by me.]

science march banner.jpg

According to the BBC, “thousands of people” joined the march, the first of its kind taking place on the annual Earth Day and organised around the world. I think the event probably got part of its boost from the Women’s Marches that took place on January 21st, the day after Donald Trump’s inauguration. Certainly, the protesters on both occasions had much in common.

destroy the patriarchy, not the planet

One of the most notable differences between the two events, however, was that this second protest was certainly smaller and also much quieter. I suppose it’s true that scientists – and yes, the marchers were mainly scientists and their relatives, partners and close friends – are a little bit shy and socially awkward. Amongst the stewards, one was trying to get the following chant off the ground, with little success, “Scientists are good at generating questions, not so good at slogans”…

french embassy

Here’s a blurry Li in the foreground, with a sharp French embassy in the background. Walking by I couldn’t help but send what’s known as a “Stoßgebet” in German to the high heavens; roughly translates as a quick (secular) prayer. For now we can breathe a brief sigh of relief after the first round of the presidential elections. Hopefully Europe, science and European Research Council funding will be able to continue to prosper.

knowledge trumps ignorance

Speaking of Trump, the March for Science event emanated from Washington DC, where it started as a protest against fake news, alternative facts and a world in which experts are regarded as worthy of derision. Honestly, as with the Women’s March, I don’t know and can’t tell how much impact marches like these actually have in politics, but as a start there was significant media coverage. Even Buzzfeed compiled its list of top banners and slogans (some scientists do have a sense of humour). My personal favourite was this one, of course.

big brains

I do know that within three months I went to two marches, the first two of my life. Ideally, I won’t have to go to any more and will be able to spend my Saturdays in the lab, where a diligent PhD student should be (and where I know some of my colleagues were). Lastly, let’s give reason, described by Wikipedia as being “the capacity for consciously making sense of things, applying logic, establishing and verifying facts, and changing or justifying practices, institutions, and beliefs based on new or existing information”, a big thumbs up.

reason

Back to Cell Biology

Because you’ve just gotta love cells. And because this post is about a publication in The Journal of Cell Biology, published by the Rockefeller University Press. In the summer of 2014 I spent almost three months doing an undergraduate research programme at Cold Spring Harbor Laboratory in the lab of Lloyd Trotman and under the everyday supervision of Dawid G. Nowak. I mainly helped Dawid establish the CRISPR/Cas9 method in the lab to study several types of cancers, including lung and prostate cancer. The first story, in which we used CRISPR to knockout a potent oncogene called Myc, was published almost two years ago (Nowak et al, 2015). Now Dawid is the co-first author on a new paper studying a tumour suppressor protein called PTEN (Chen, Nowak, … Wang, … et al, 2017).

Here is an eLife-style digest of the manuscript. Tumours usually evolve when cells gain the function of so-called oncogenes and lose the function of one or more so-called tumour suppressor genes. One of the most frequently deleted or down-regulated tumour suppressors is a protein called PTEN. Some cancer types, including some types of lung and prostate cancer, do not always delete the two gene copies coding for the PTEN protein, but the levels of PTEN protein in those cancer cells is still kept low. Therefore we wanted to find out which pathways in cancer cells lower the PTEN protein levels. Knowing about this regulation could lead to the development of new therapies that aim at stabilising PTEN protein.

First, we used both mouse and human cancer cell lines to investigate the movement of PTEN between the cytoplasm and the nucleus. We hypothesised that PTEN might be protected from being degraded in the nucleus, since the enzymes that break proteins down are generally found in the cytoplasm. Biochemical experiments showed that PTEN was moved into the nucleus by a protein called importin-11. Next, and this is the experiment I performed, we deleted importin-11 using CRISPR/Cas9 and saw that PTEN abundance decreased, while active/phosphorylated Akt, an oncogene, increased:

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Western blot showing CRISPR/Cas9 deletion of importin-11 in human prostate cancer cell lines – copied directly from Fig. 2 of Chen, Nowak et al, 2017

Further experiments conducted in the cell lines supported the following model, in which PTEN is shuttled into the nucleus by importin-11 where it is protected from degradation by the ubiquitin ligase system:

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Model of PTEN shuttling: when importin-11 is present PTEN can “hide” in the nucleus (left), but when importin-11 is deleted/not functioning, PTEN accumulates in the cytoplasm where it can be targeted for degradation – copied directly from Fig. 4 of Chen, Nowak et al, 2017

Next we wanted to know whether this mechanism of keeping levels of PTEN low is also important for preventing tumours. When importin-11 was experimentally down-regulated in mice (the gene for importin-11 was not completely deleted but its mutation is said to be “hypomorphic”), the mice developed and eventually died from lung cancers, unlike the healthy control mice:

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Lesion-free survival curve of importin-11 mutant (red) versus control (black) mice – copied directly from Fig. 5 of Chen, Nowak et al, 2017

Similar results were also obtained for prostate tumours in mice. Lastly, we analysed publicly available data of human prostate cancer patients. Low levels of importin-11 (either by genetic deletion or low gene expression) correlated with higher rates of tumour recurrence, suggesting that importin-11 also acts as a tumour suppressor in some types of human cancer. Future experiments may involve conducting more sophisticated mouse experiments in which importin-11 is deleted in specific organs, together with the activation of known oncogenes. This work may also lead to studies that try to find ways of stabilising PTEN protein.


So that’s it. Publication number three! But I want to end on a slightly more philosophical/political note. Dawid, one of the two first authors, taught me a lot during that summer programme, has been supportive ever since, and I enjoy keeping in touch with him. At the moment he is looking for an independent research position – he is enthusiastic about science and very driven. He’s had interviews all over the place, both in Europe and North America. However, Dawid is Polish and is now having to re-think his options since neither the UK nor the USA seem particularly appealing places for him anymore. We live in a crazy world but I hope this won’t stop him from getting the lab he deserves, in the most tolerant place possible.


References:

Chen M, Nowak DG, Narula N, Robinson B, Watrud K, Ambrico A, Herzka TM, Zeeman ME, Minderer M, Zheng W, Ebbesen SH, Plafker KS, Stahlhut C, Wang VMY, Wills L, Nasar A, Castillo-Martin M, Cordon-Cardo C, Wilkinson JE, Powers S et al. (2017) The nuclear transport receptor Importin-11 is a tumor suppressor that maintains PTEN protein. The Journal of Cell Biology DOI: 10.1083/jcb.201604025

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