CRISPR Digest #11

By this point my interest in CRISPR research is apparently well known to the people around me: a friend shared a research article with me on Facebook by Kaminski et al. (2016) published earlier this month in the journal Scientific Reports. In this paper the researchers used the CRISPR/Cas9 genome-editing technology to eliminate the human immunodeficiency virus (HIV) genome from infected cells.

HIV is the causative agent of acquired immune deficiency syndrome (AIDS): the virus infects some of the so-called T cells in our immune system and integrates its genetic material into the DNA of the host cell. The infection causes a lot of T cells to die and eventually – over a period of years or often even decades – this makes the immune system less and less efficient. Therefore the causes of death of AIDS patients are usually infections, which would normally be fought off by a healthy immune system, or rare types of tumours. Currently there are efficient retroviral therapies available to treat HIV infection. However, these therapies do not remove the virus DNA from the patient, rather they keep the virus at bay (it is said to be “latent”). Therefore the treatment is usually lifelong and expensive.

Kaminski et al. mainly used a human cell line to test whether they could design guide RNAs that would specifically guide the Cas9 protein to the DNA sequences at either end of the HIV genome. The cell line is called 2D10 and has been well characterised and has a single HIV genome inserted at a known location, making it a good model to test their experimental tools. Since CRISPR is such a ubiquitous tool in the lab already a lot of the paper actually focusses on making sure that cutting out the HIV genome – which they manage successfully – does not have any unintended consequences. In particular, the researchers checked that Cas9 does not introduce mutations elsewhere in the host genome.

Having established these controls Kaminski et al. then go on to show that 2D10 cells with Cas9 and the guide RNAs (gRNAs) are less susceptible to a new HIV infection. To further test their system the researchers used human T cells from healthy individuals to show that these cells can also be made more resistant to infection when given the Cas9/gRNAs. Lastly, the paper shows that the technique can also target HIV DNA in human T cells from infected patients. However, here the Cas9 was not able to entirely excise the HIV genomes. Partly this can be attributed to the fact that human cells are much more heterogeneous than the 2D10 cell line: the virus will have integrated at different sites in the host genome in different T cells and there may also be several integration sites per cell.

This is an impressive study and a good step towards being able to treat patients with HIV using genome-editing technology, but there are still some shortcomings. To me one of the main problems seems to be the way in which the Cas9 and guide RNAs are delivered into the infected T cells: often this is done by putting the DNA that codes for the Cas9 protein and the gRNAs into a lentivirus, which belongs to the same group of viruses as HIV itself. The lentivirus would therefore itself integrate into the genome of the host cell and this might cause problems in itself, for example, by disrupting important genes. Furthermore, and the authors allude to this, not all HIV genomes are exactly the same and so for each patient one might have to design individual gRNAs.


Since we are on the topic of HIV/AIDS I would like to mention something another friend has brought to my attention. Some countries, such as the USA, Canada and France, have programmes to make so-called pre-exposure prophylaxis (PrEP) available to people who are HIV-negative but at high risk of contracting the infection. The National Health Service in England has recently released a statement explaining that it will no longer pursue this avenue although the once-daily pill has been shown to decrease the relative risk of becoming infected by over 90% in men who have sex with men (see, for example, Grant et al., 2010). The National AIDS Trust has therefore started a campaign for people to write to their local MPs so that this issue can be raised in parliament. And I did just that right now and realised that my MP is none other than Jeremy Corbyn.


On a slightly more upbeat note, here is a wallpaper design by Nature for their special CRISPR issue (downloaded directly from their website):

crispr wallpaper

And regarding the CRISPR patent dispute, there was a good News & Views article on the topic a few weeks ago and the take-home message is that it will probably take several years for it to be decided.

Lastly, happy Easter to all those who celebrate it in one way or another. Instead of (chocolate) eggs I will share with you a slightly abstract art image that I inadvertently took on the microscope a couple of months ago. With a little bit of imagination the organoids could be mistaken for Easter eggs.

organoids


References:

Grant  RM, Lama  JR, Anderson  PL, McMahan  V, Liu  AY, Vargas  L, Goicochea  P, Casapía  M, Guanira-Carranza  JV, Ramirez-Cardich  ME, Montoya-Herrera  O, Fernández  T, Veloso  VG, Buchbinder  SP, Chariyalertsak  S, Schechter  M, Bekker  L-G, Mayer  KH, Kallás  EG, Amico  KR, Mulligan  K, Bushman  LR, Hance  RJ, Ganoza  C, Defechereux  P, Postle  B, Wang  F, McConnell  JJ, Zheng  J-H, Lee  J, Rooney  JF, Jaffe  HS, Martinez  AI, Burns  DN, Glidden  DV (2010) Preexposure Chemoprophylaxis for HIV Prevention in Men Who Have Sex with Men. New England Journal of Medicine 363: 2587-2599

Kaminski R, Chen Y, Fischer T, Tedaldi E, Napoli A, Zhang Y, Karn J, Hu W, Khalili K (2016) Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing. Scientific Reports 6: 22555

Of mimiviruses and making progress

It’s been so long since I’ve written anything about CRISPR that I feel completely rusty. Luckily, I spotted some interesting new research on an “immune system” found in giant viruses called mimiviruses. Levasseur et al. (2016) propose that mimiviruses – large viruses that can be seen with a light microscope and have a genome that is bigger than that of some bacteria – can defend themselves against so-called virophages. In analogy to the viruses that infect animals, bacteria can be infected by viruses called bacteriophages, and mimiviruses can be infected by virophages. Although generally viruses are regarded as non-living these giant viruses do possess some genes that code for proteins that help build the virus. Normally, viruses hijack the infected host cell’s machinery to replicate and are completely dependent on the host cell. Only last year a group reported (Ekeberg et al., 2015) using high power X-rays to study a single virus and being able to look inside the particle. There is a nice 3D reconstruction video in the Nature News & Views article.

One of the virophages that infects some mimivirus lineages, but not others, is called Zamilon. Levasseur et al. guessed that mimiviruses  might incorporate stretches of DNA from Zamilon and use them to recognise infecting virophages. This would be in complete analogy to many bacteria and archaea that protect themselves from their attackers using CRISPR – clustered regularly interspaced short palindromic repeats. The DNA “spacers” correspond to the sequences found in the invading DNA  of bacteriophages, for example. To test their idea, Levasseur et al. sequenced the genomes of 45 mimivirus strains and did indeed find a repeating region that corresponded to Zamilon DNA. They called it MIMIVIRE, for mimivirus virophage resistance element (the figure is copied directly from the paper):

Screen Shot 2016-03-05 at 21.30.06

Schematic of the mimivirus MIMIVIRE locus – copied directly from Levasseur et al., 2016

Nearby in the mimivirus genome the researchers also found (Cas-like) genes that might be involved in this defence mechanism: when those genes were genetically silenced the viruses that were normally resistant became sensitive to Zamilon infection. Furthermore, these Cas-like genes code for proteins that can bind and modify DNA and are therefore perfect candidates for destroying invading DNA. More experiments will need to be carried out to show exactly how the MIMIVIRE system works, but it is likely to be different from the CRISPR/Cas system. However, it is interesting to see how immune systems are pervasive in all domains of life (and almost life) and although I am no evolutionary biologist I think this may be an example of convergent evolution. [Thoughts on this would be appreciated!]


To end today’s post on a personal positive note: this year’s birthday present was being able to image the cells I am studying by electron microscopy. Electron microscopes are, in principle, similar to light microscopes, which most of us have used at school, except that they utilise electron beams instead of light rays as the source of illumination. Since electron beams have a much shorter wavelength (i.e. are higher in energy) than visible light they can visualise objects with much higher resolution, making it possible to get sharp images at greater magnification. Some of the images we – the extremely knowledgeable and helpful scientists at the electron microscopy facility in our institute and I – took were magnified 60,000 times!

Although I can’t share those images at the moment, here is a transmission electron micrograph of a normal pancreatic ductal cell (copied directly from here):

wt ductal cell

Pancreatic ductal cell

In the centre of the cell is the large nucleus, clearly defined by its double membrane. The darker patches within the nucleus are parts of the DNA that are more tightly compacted than others. In the cytoplasm (the area that isn’t the nucleus) there are some mitochondria, where a lot of the cell’s metabolism takes place, and so-called endocytic vesicles, membrane-bound compartments that are involved in recycling and housekeeping. At the top left are microvilli, short protrusions of the cell into pancreatic duct. Although I’m not sure I can spot it here, the images we took also revealed the rough endoplasmic reticulum where ribosomes sit and produce proteins. Can you believe I saw ribosomes?!

Lastly, and this goes out mainly to all the other PhD students, I’ve recently been finding it helpful and refreshing to relish the feeling that, as a student, there is always so much more to learn. To really bask in the glory of one’s own ignorance and then pester and listen to more knowledgeable and more experienced people to learn something new. For example, you can plan a decent experiment that has the proper controls and would give you some useful information. Then you tell your supervisor about it and s/he suggests doing that little extra something that suddenly turns the experiment into real science. On the one hand, I find these occurrences humbling. But, on the other hand, they are also exciting because I’m finding it easier and easier to recognise what makes a really good experiment and I can see, still beyond my reach but tantalisingly close, the potential of making that last mental leap myself. Needless to say that doesn’t at all mean that an experiment will technically work…


References:

Ekeberg T, Svenda M, Abergel C, Maia FRNC, Seltzer V, Claverie J-M, Hantke M, Jönsson O, Nettelblad C, van der Schot G, Liang M, DePonte DP, Barty A, Seibert MM, Iwan B, Andersson I, Loh ND, Martin AV, Chapman H, Bostedt C et al. (2015) Three-Dimensional Reconstruction of the Giant Mimivirus Particle with an X-Ray Free-Electron Laser. Physical Review Letters 114: 098102

Levasseur A, Bekliz M, Chabrière E, Pontarotti P, La Scola B, Raoult D (2016) MIMIVIRE is a defence system in mimivirus that confers resistance to virophage. Nature advance online publication