CRISPR Digest #4

Lent Term at Cambridge has officially ended and with it the last day of work in the lab and my last ever undergraduate lectures and seminars. Now begins the period of analysing results and trying to formulate a coherent dissertation, but that should be the subject of a different entry.

The last so-called “scientific methodology” workshop we had this term was entitled CRISPR/Cas9 – you can imagine my excitement about this, especially because it turned out that I learnt at least three new facts in that session (quite a lot more than can be said about some of the cancer lectures, which were more or a less a repetition from last year).

1. The functions of the so-called protospacer adjacent motif (PAM), a short DNA sequence present in the target DNA, just next to the cleavage site, but not the guide RNA; for the widely used Streptococcus pyogenes type II CRISPR/Cas9 system it is NGG:
   1. The PAM possibly provides a means of simplifying the search for the correct target sequence.
   2. Binding of Cas9 may be initiated at the PAM sequence from where the transition to active DNA cleavage may take place.
   3. Most importantly – and this is something I hadn’t at all thought of before – the PAM can provide a mechanism for the discrimination between self and non-self, a fundamental feature of all immune systems. CRISPR systems evolved as adaptive immune systems in bacteria and archaea, targeting invading DNA from bacteriophages (viruses that infect bacteria) and plasmids. The host genome incorporates stretches of the invading DNA and then expresses that as guide RNAs, which in turn direct the Cas nucleases against the invading DNA. But of course this means that the guide RNAs also interact with the DNA that was incorporated in the host genome in the first place, which would lead to cleavage of host DNA – quite the opposite of the desired outcome. Therefore, the host sequence does not contain the necessary PAM sequences so that the Cas nucleases cannot attack their own host! [See, for example, Mali et al. (2013) or Marraffini & Sontheimer (2010).]
      

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2. Novel methods of regulating the activity of Cas9 variants so that gene expression can be fine-tuned in target cells of interest:
   1. The first of two papers discussed (Nihongaki et al. (2015)) combined the CRISPR system with optogenetics, a tool originally developed by neuroscientists to selectively stimulate or suppress the activity of specific neurons in the brain: in this system Cas9 is catalytically inactive (dCas9; cannot cleave DNA), but is fused to a protein called CIB1. Additionally, a transcriptional activator/repressor is fused to a protein domain of CRY2. Upon irradiation with blue light CIB1 and CRY2 interact, thus bringing the transcriptional regulator in proximity with dCas9, which has been targeted to a region of interest by a guide RNA. This allows controlled expression of genes of interest from their endogenous loci (image copied from their paper):

      

   2. In the second paper by Zetsche et al. (2015) they engineer a split Cas9, in which each half of the protein is fused to a domain from the protein mTOR (normally involved in regulating cell growth). These domains dimerise upon addition of the drug rapamycin, thus activating Cas9 because the two halves are brought together again. This approach allows temporal control of Cas9 activity and can also be used with dCas9 to regulate gene expression. However, the concern I have with the method is that rapamycin is a drug that inhibits mTOR and, to my knowledge, the paper does not address the question of whether the drug treatment in itself is detrimental to the cells.
      

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3. Lastly, we were introduced to a new, CRISPR-based technique that I had not encountered before: enChIP – engineered DNA-binding molecule ChIP (Fujita & Fujii (2013)). ChIP stands for chromatin immunoprecipitation and in its basic form relies on specific antibodies with which proteins of interest can be “pulled out” together with other proteins and DNA they are interacting with. For example, this technique can be used to find out where in the genome protein X binds – does it bind the promoter regions of certain genes? Does it bind to a specific DNA sequence repeatedly? Etc.
   In enChIP, dCas9 is fused to a short peptide known as a FLAG tag, against which very good antibodies exist. This fusion is then targeted to a DNA sequence of interest (or very close to a sequence of interest) where the protein complexes formed are crosslinked before being “pulled out” with the anti-FLAG antibody. The complexes are subsequently subjected to mass spectrometry, which can resolve the constituent parts of the complex. In a way, therefore, enChIP can provide complementary information to standard ChIP: in the former one is looking for proteins that bind to specific DNA sequences, whereas in the latter one is looking for DNA sequences bound by specific proteins.

Hopefully this digest will keep you all satiated with information on the latest CRISPR news for a while. Now: time to dissertate (yes, the OED assures me that this is indeed a word, if somewhat “unusual”).

References:

Fujita T, Fujii H (2013) Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochemical and Biophysical Research Communications 439: 132-136

Mali P, Esvelt KM, Church GM (2013) Cas9 as a versatile tool for engineering biology. Nature Methods 10: 957-963

Marraffini LA, Sontheimer EJ (2010) Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463: 568-571

Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M (2015) CRISPR-Cas9-based Photoactivatable Transcription System. Chemistry & Biology 22: 169-174

Zetsche B, Volz SE, Zhang F (2015) A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotech 33: 139-142

In silico versus in vitro #2

There is no adequate excuse for not keeping up with regular blog entries. Arguably, working in the lab from 9 a.m. to 8.30 p.m. – in a mad rush to collect some data before the lab meeting next week – in addition to preparing for seminars and the imminent badminton match against Oxford help to explain the recent scarcity of cell/science-related news. Especially when a statistically significant number of experiments in the lab end up looking like this Western blot on the right (image copied from here):

However, on the more positive side of things I have learnt a couple more cell/molecular biology techniques since I started working in the lab several weeks ago. Beforehand I only knew about the theory of these methods and how to interpret results, but not how to actually carry out the experiments:

• Immunohistochemistry (IHC): this technique allows specific staining of (mouse) tissue sections. One can, for example, compare specific markers of proliferation or cell death in healthy skin versus tumours/melanoma lesions.
• Quantitative reverse transcription polymerase chain reaction (qRT-PCR): this allows analysis of gene expression (specifically transcription) in tumour samples or cultured cells under different conditions. For example, I have been comparing the expression of certain transcription factors in cells that are sensitive to a melanoma drug versus cells that have acquired resistance to that drug.
• Cell cycle analysis by fluorescence-activated cell sorting (FACS): lastly, this method is used to quantify the proportion of cells within a population that are actively dividing, non-dividing or dead. To do this, the amount of DNA in each cell is stained using a dye such as propidium iodide, or newly synthesised DNA can be labelled using bromodeoxyuridine.

Now on to the simple task of analysing the data…

In silico versus in vitro

For the past five days at least I have felt bad for not updating my blog with some fact about CRISPR or the latest controversy concerning “cancer stem cells”. The reason for this involuntary hiatus is, of course, lab work. All the lab work. All the time. How should I put this; there are distinct benefits to working in silico: leaving the lab/office whenever is convenient, being able to continue work at home, and actually getting home on time for dinner.

Here I am, genuinely happy to be doing work in cell culture (isn’t the dark blue of that lab coat excellent?):

And then running samples in pre-cast 20-well gels ready to do a Western blot after having treated melanoma cells with a plethora of small molecule inhibitors:

But then disaster had to strike. It was all going too well. Due to the bubbles rising from the electrodes in the above picture I didn’t have a clear view of the second gel running behind it. What a disappointment:

The sad smiley face accurately represents my emotions concerning this gel. Well, at least I know what I’m doing tomorrow.

So I’ll end with a mini CRISPR update: Tsai et al. (2014) developed a new method last year to reduce non-specific cleavage of DNA during genome-editing. They require expression of RNA-guided FokI nucleases, which also cleave DNA, but are only active when dimeric. Each single FokI molecule is guided to its target by a guide RNA, but only when two guide RNAs each bring a FokI to the desired locus do the enzymes become active. This drastically reduces off-target effects because both the sequence and spacing has to be correct. The original CRISPR/Cas9 system has considerable off-target effects, as shown by Lin et al. (2014), for example.

Reference:

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

Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotech 32: 569-576

Seminars with Sirs

In addition to normal lectures and lab/project work, the M.Sci. biochemistry course here at Cambridge also includes weekly seminars, which focus either on a set of landmark papers or on a particular methodology. The general idea of these seminars is probably “to encourage students to think, to learn, and to think about learning, so that they ultimately develop the skill—and courage—to train themselves” (Raman (2015)). Raman argues in the eLife article that understanding past research in its historical context, whose results and implications are now taken for granted, is a key step to being able to come up with interesting questions and the appropriate experimental approaches to tackle these. Maybe in a few years we’ll know whether reading and discussing these landmark papers actually had this desired effect.

Two weeks ago the seminar was entitled “Greatwall and the control of mitosis” and was held by this charming fellow:

He is none other than (Professor Sir) Tim Hunt, who won a Nobel prize for Physiology/Medicine in 2001 together with Paul Nurse and Lee Hartwell for their “discoveries of key regulators of the cell cycle”. I would wager that the foundation experiments leading to this prize are taught in all biology undergraduate courses and so the seminar was not actually about these, but rather on the follow-up experiments conducted by Tim Hunt and his lab. In particular, the seminar was about the intricacies of cell cycle regulation by proteins called phosphatases. Phosphatases are enzymes that remove phosphate groups from other proteins and thus catalyse the opposite reaction of protein kinases, which add phosphates to proteins, usually at the amino acids serine, threonine or tyrosine. Some of what we discussed was summarised by Mochida & Hunt (2012), but the exciting and interesting parts of the seminar actually consisted of listening to Tim Hunt explain which experiments he agreed with and why, and perhaps more entertainingly, which experiments he does not believe and why.

Then a week later the seminar was hosted by John Walker:

(Professor Sir) John Walker – surprise, surprise – also won a Nobel prize (Chemistry, 1997), together with Paul Boyer for “their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)”. [Incidentally, John Walker seems quite proud of being a knight of the British Empire: I caught a glimpse of the inside of the case for his glasses, “Sir John Walker + telephone number”.]

In this seminar we also briefly recapped the basics of ATP production by mitochondria, but again this is something we covered in first and second year. However, we then discussed the landmark paper (Abrahams et al. (1994)) describing the structural features of the enzyme ATP synthase that catalyses the production of ATP from ADP and phosphate. Incidentally, that paper was dedicated to Max Perutz for his 80th birthday, since he was involved in discussing this research at the MRC Laboratory of Molecular Biology. Subsequently, we moved on to more current topics relating to ATP synthase, such as its possible involvement in the formation of the mitochondrial permeability transition pore (Giorgio et al. (2013)).

Interestingly, although Hunt and Walker are of course entirely different people, there were two striking similarities between them: firstly, both of them are still active researchers who clearly are still excited by science and their experiments. Secondly, both are embracing new techniques and technologies, which were not available when they started out as scientists. For example, Walker and his group use molecular dynamics simulations (quantum mechanics and computation) as well as cryo-electron microscopy to study ATP synthase. Hunt also uses computational modelling to gain more insights into the complex networks regulating progression through the cell cycle, and Paul Nurse, who used to be a “simple” geneticist, has now essentially become a systems biologist. Hard work, joy at doing science and being receptive to new technologies all seem to be hallmarks of good researchers – best to bear this in mind.

References:

Abrahams JP, Leslie AGW, Lutter R, Walker JE (1994) Structure at 2.8-angstrom resolution of F1-ATPase from bovine heart mitochondria. Nature 370: 621-628

Giorgio V, von Stockum S, Antoniel M, Fabbro A, Fogolari F, Forte M, Glick GD, Petronilli V, Zoratti M, Szabo I, Lippe G, Bernardi P (2013) Dimers of mitochondrial ATP synthase form the permeability transition pore. Proceedings of the National Academy of Sciences of the United States of America 110: 5887-5892

Mochida S, Hunt T (2012) Protein phosphatases and their regulation in the control of mitosis. Embo Reports 13: 197-203

Raman IM (2015) Teaching for the future, Vol. 4.; eLife 2015;4:e05846

PhD Interview for the Francis Crick Institute!

Despite being funded as a Cancer Research UK charity, the London Research Institute (LRI) went to considerable lengths to ensure that we interviewees were comfortable during our three-day visit to London and the institute. Firstly, our travel expenses – ranging from short intra-England train journeys to flights from across Europe and North America – were covered, as well as our accommodation at a hotel overlooking Russell Square at the heart of Bloomsbury:

The first day was probably the most strenuous. First we listened to introductory talks given by the LRI Academic Director and the LRI’s Deputy Director who, incidentally, also quoted Donald Rumsfeld about the unknown unknowns just like at the departmental research day. Furthermore, as part of my destressing strategy I took a walk around the area during one of the breaks, inevitably stumbled into a bookshop and found this:

The rest of the first day was filled by talks given by each of the recruiting group leaders. Eighteen times ten minutes of concentration. After that we got the chance to speak to those principal investigators (PIs) we were interested in. Lastly, we had dinner with the PIs and some of their students. And although all of this was not part of the “official” assessment procedure I think it was important to be making a good impression throughout, and therefore by the end of this first day most of us felt exhausted.

The official panel interviews were scheduled for the second day. We each had to give a presentation of a research project we were involved in, as well as a critique of a research paper. We were then asked some questions on these presentations and also had the usual questions hurtled at us, “Why do you want to do a PhD? Why do you want to do it at the LRI? What are your long-term goals?” Etc.

We were also privy to a tour of the LRI building at Lincoln’s Inn Fields. During the introductory talks they emphasised how great the facilities – DNA sequencing, flow cytometry and microscopy among others – at the LRI are. I was skeptical at first, but the tour was convincing, especially considering that probably not so much money is being invested in the upkeep of this building due to the move of the LRI into the Francis Crick Institute in 2016. At the Crick of course everything will be even better, as they didn’t fail to mention at every possible opportunity.

On the second day we had dinner together with the lab members of the recruiting labs, but without the PIs who were busy trying to work out who to invite for the third day on which one-on-one interviews would be held. We were certainly more relaxed this evening. However, the next morning between 7.15 and 8.00 am we had to come down into the reception area of the hotel to pick up a letter informing us whether we had been invited for the third and final day. It was irrational to be nervous because at this stage there was absolutely nothing to be done about the situation. Nevertheless, I, and probably many others, had difficulty sleeping that night.

Luckily, I was invited back to speak to three group leaders: Axel Behrens, Victoria Sanz-Moreno with Ilaria Malanchi, and Caroline Hill. In these sessions it became clear that I would want to work either with Axel on pancreatic cancer or with Victoria and Ilaria on melanoma. The third project was more focussed on neurodevelopment, which is interesting but my gut feeling told me to veer away from it simply because I have a stronger background in cancer biology.

At the end of the third day we had to hand in a preference list, and then all there was left to do was to go back to Cambridge and wait. But the waiting was mainly a formality since it had become clear during the day that Axel Behrens’ lab was going to make me an offer I couldn’t refuse. I am extremely excited! London, here I come!