CRISPR Digest #9

The Economist magazine had a field day in their issue of October 17th 2015: in their article, “No pig in a poke” – with one of the subtitles being “Go the whole hog” – they describe the latest findings by Yang et al. (2015) in which they used CRISPR/Cas9 genome-engineering technology to remove potentially contagious virus sequences from pig genomes. These DNA sequences are called porcine endogenous retroviruses (PERVs for short) and could move into the human genome if, for example, a pig heart were transplanted into a human. In addition to the problem of immune rejection of the organ, therefore, there is also the risk of infection with porcine viruses/DNA sequences. The Economist concluded that, “The existence of PERVs, then, has been one of the main obstacles to transplanting pig organs into people”. In this proof-of-concept paper by Yang et al. the scientists managed to remove DNA sequences necessary for the PERVs to move from the pig genome into the human genome. However, this was done in cell culture and not in whole pigs, “but this is still a striking result” despite the fact that “a single paper does not a new medical procedure make”. [I did not realise quite how poetic the writers at The Economist can be.]

In other news, more basic research on CRISPR has revealed that the Cas9 enzyme – the protein that makes the cut in DNA – of a bacterial species called Neisseria meningitidis can also cut single strands of DNA (Zhang et al., 2015), in addition to its expected role of cutting double strands of DNA. This is of interest because it allows a wider range of applications, especially considering that N. meningitidis Cas9 is considerably smaller, and therefore easier to handle from an experimental point of view, than Streptococcus pyogenes Cas9, which is currently the most commonly used Cas9 version.

While many research groups are working on understanding new mechanisms of gene editing and how various Cas9 variants function in detail, the group of Jennifer Doudna published a paper in Nature this week outlining how E. coli acquire foreign DNA and integrate it into their genomes’ CRISPR arrays (Nuñez et al., 2015). They studied the crystal structure of the complex of the Cas1 and Cas2 proteins, which are well conserved between bacterial species, and the DNA to be integrated:

X-ray crystal structure of E. coli Cas1-Cas2 in complex with double-stranded DNA - image copied directly from Nuñez et al., 2015

X-ray crystal structure of E. coli Cas1-Cas2 (blue/green and yellow) in complex with double-stranded DNA (red) – image copied directly from Nuñez et al., 2015

In particular, Nuñez et al. found that the Cas1-Cas2 complex acts as a molecular ruler to determine the length of DNA that is incorporated into the E. coli genome. Furthermore, they demonstrate that Cas1 can pry open the ends of the DNA, as seen in the image above, so that Cas2 – which is catalytically active – can gain access to the reactive ends of the DNA. So here is yet another example of how the structure and function of proteins are intricately connected and often knowledge of one can enhance knowledge of the other.

References:

Nuñez JK, Harrington LB, Kranzusch PJ, Engelman AN, Doudna JA (2015) Foreign DNA capture during CRISPR–Cas adaptive immunity. Nature advance online publication

Yang L, Güell M, Niu D, George H, Lesha E, Grishin D, Aach J, Shrock E, Xu W, Poci J, Cortazio R, Wilkinson RA, Fishman JA, Church G (2015) Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science

Zhang Y, Rajan R, Seifert HS, Mondragón A, Sontheimer Erik J (2015) DNase H Activity of Neisseria meningitidis Cas9. Molecular Cell 60: 242-255

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A PhD Student for a Month

After the week of induction I have now spent three weeks in the lab. I have cells to look after, so that is most definitely a good thing. Mainly I am trying to figure out what kind of experiments to do that will actually give some sort of result and that is of course a lot harder than most papers will have you think. Until now I have just been finding my bearings in the lab (e.g. where do you keep the protease inhibitors? What? You freeze down your protein ladders/markers in the -80 °C freezer? Why can’t I find the antibodies in the boxes they’re meant to be in according to the Excel sheet? Etc.)

Luckily, people in the lab are all friendly and willing to help out. Despite that I still managed to crush a bottle in the centrifuge, by spinning it at 10,000 times the gravitational force. Turns out that it doesn’t need to be spun that quickly and quite evidently can’t take those forces. And this was already the second try. The first time I actually managed to rupture the bottle, losing some of my sample…

thumb_IMG_0728_1024

Then of course there are the always unpredictable Western blots, as already alluded to earlier, in which one can investigate whether particular cells express certain proteins of interest. Ideally, this sort of experiment should yield a straightforward answer (more or less), as on the left of the following picture. But who even knows what happened on the right.

Screen Shot 2015-10-18 at 10.19.53

Slotted between these attempts at lab work I’ve attended training to use the fancy flow cytometers, which can analyse single cells and even sort them according to different properties, and confocal microscopes. And so, let the next week of lab commence!

CRISPR Digest #8

Time for an update. Over the summer the Innovative Genomics Initiative (IGI) hosted a one-week CRISPR workshop where various speakers held lectures addressing how they are using the technology in their research and how they propose to proceed with it. IGI made these lectures available here and had, among others, Jennifer Doudna speak about her group’s work on the mechanism of CRISPR/Cas9:

More recently and perhaps a little more excitingly, researchers led by Feng Zhang at the Broad Institute in Cambridge (USA) have published a paper (Zetsche et al., 2015) describing a new protein – called Cpf1 – from the bacterial species Acidaminococcus and Lachnospiraceae that can also cut DNA, like the now widely used Cas9 protein. However, it acts slightly differently from Cas9 in that it only needs a single guide RNA to find its target DNA and importantly, it cuts the DNA in a way that leaves so-called “sticky ends”. These cut sites with “staggered” ends allow much more controlled insertion of new DNA into the cut site. This, in turn, means that cells can now be more easily made to express engineered proteins with specific mutations, for example, or corrected versions of proteins. Although this was possible using the original CRISPR/Cas9 system it was not very efficient. However, pictures supposedly say more than a thousand words so here is their “graphical abstract”…

Graphical abstract of how Cpf1 can target DNA with the help of a single guide RNA - copied directly from Zetsche et al., 2015

Graphical abstract of how Cpf1 can target DNA with the help of a single guide RNA – copied directly from Zetsche et al., 2015

Lastly, I just want to mention that I have noticed that the original two pioneers of CRISPR technology, Emmanuelle Charpentier and Jennifer Doudna and their research teams, are sticking to basic research. That is to say they study the exact mechanisms by which CRISPR/Cas9 works, elucidate the atomic structures of these protein-RNA-DNA complexes and are still looking for other related systems to better understand how bacteria protect themselves from infections by plasmids (circular bits of DNA) and bacteriophages (viruses that infect bacteria). Despite the high-profile patent case they are involved in and all the other labs around the world that are now using this technology in various applications, such as gene therapy or plant engineering, they remain focussed on simply increasing our knowledge of nature, which I find laudable.

Reference:

Zetsche B, Gootenberg Jonathan S, Abudayyeh Omar O, Slaymaker Ian M, Makarova Kira S, Essletzbichler P, Volz Sara E, Joung J, van der Oost J, Regev A, Koonin Eugene V, Zhang F (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell (in press).