CRISPR Digest #13

It’s time to go back to some of the basic biology behind the whole CRISPR gene-editing hype. This week Cell and Molecular Cell published two nice papers on the why and how of CRISPR.

In one of my earliest posts on this blog, CRISPR Craze, I gave a brief overview of how CRISPR works in prokaryotes. I’ll reiterate here: bacteria and archaea have evolved a response against invading pathogens, often bacteriophages (viruses that infect bacteria), which has been compared to our mammalian immune system. In essence, CRISPR allows bacteria to recognise when a pathogen, and specifically its DNA, is infecting the cell again. During the first round of infection the bacterium incorporates parts of the pathogen’s DNA in its own genome and therefore keeps a record or memory of that invader. Then, during a second round of infection, the DNA can be transcribed into RNA by the bacterium, which is used as a “guide” to detect the invading DNA (since the RNA and DNA will be complementary). Additionally, the guiding RNA will bring/guide one (or several, depending on the exact type of system) so-called CRISPR-associated protein (Cas) to the invading DNA. The Cas protein(s) is then responsible for cleaving the pathogen’s DNA and thus thwarting the infection. CRISPR in a nutshell. Easy.

If you think about it, the findings from Pawluk et al., 2016 will not come as a surprise. First, bacteriophages infect bacteria. Second, bacteria evolve an intricate mechanism to defend themselves against the viruses and other, potentially harmful “mobile genetic elements”. So third – the logical conclusion – bacteriophages find a way to shut down the CRISPR defence. Pawluk et al. found that the Cas9 protein in the bacterial species Neisseria meningitidis can be inhibited by phage anti-CRISPR proteins:

anti-crispr

Schematic representation of the anti-CRISPR system, which can also be used in mammalian gene-editing system – image copied directly from Pawluk et al, 2016

In particular, Pawluk et al. discovered three acr genes in N. meningitidis, which code for the Acr proteins. The acr genes are incorporated into the bacterial genome but originally came from bacteriophages or mobile genetic elements. Biochemical experiments showed that the Acr proteins can bind directly to Cas9 and stop it from cutting DNA. Lastly, Pawluk et al. demonstrated that the Acr proteins can be expressed in mammalian cells to inhibit Cas9 activity there as well. This means that future CRISPR genome-editing experiments can be fine-tuned by switching off Cas9. Being able to turn Cas9 off may be especially important for future gene therapy treatments, since preventing Cas9 from being active for too long will reduce its off-target/side effects.

The second interesting paper for this digest, Patterson et al., 2016, investigated how bacterial cells regulate when their CRISPR system is active or not. The decision to have a fully active “immune system” or not is important because it is energetically costly to have the defence mechanism in place when there is little or no threat. Patterson et al. used a species of bacteria called Serratia to examine how the density of the bacterial population influences whether CRISPR is turned on or off. Many bacterial species use a system called quorum sensing to assess whether there are many other bacterial cells nearby. For example, Serratia cells produce and secrete a small chemical (of the homoserine lactone class), which, when present in sufficient quantities, can change which genes the bacterial cells express. When the population of cells is sparse the chemical does not reach a high enough concentration to have an effect. The experiments in this paper show that at high concentrations of the small chemical, and thus at a high cell density, Serratia cells de-repress the cas genes. In other words, when there are a lot of cells in one place they collectively switch on their immune system. This makes sense: infections spread more easily among humans in crowded places and it is similar in bacterial populations. Overall, these two papers are a beautiful demonstration of how “basic” research into highly relevant and applicable technologies are still, and will continue to be, important.

Lastly, since this is the last post before Christmas and the New Year, and possibly even until we say good-bye to President Obama, let me share this resource with you:

altmetric

Screenshot from the Altmetric website listing its top 100 most-discussed journal articles of 2016

Altmetrics are “metrics and qualitative data that are complementary to traditional, citation-based metrics” and track how much and in what form scientific research is being discussed. For example, a useful but very technical paper may get many citations in the scientific literature but might not be widely talked about by people outside that field. Other new papers, that may be controversial or have wide-ranging societal implications, will also be distributed in other ways (e.g. on Twitter, Facebook, Wikipedia and on blogs). So, for your festive reading, I recommend having a browse through Altmetrics’ 100 most-discussed articles from this year. Merry Christmas!

References:

Patterson AG, Jackson SA, Taylor C, Evans GB, Salmond GPC, Przybilski R, Staals RHJ, Fineran PC Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems. Molecular Cell 64: 1102-1108

Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR (2016) Naturally Occurring Off-Switches for CRISPR-Cas9. Cell 167: 1829-1838.e1829

PhD interviews – Round 2

NHNN

The time difference between Vienna and London is only an hour, but still I had to make sure at least three times that I had really converted 10.15 (GMT) into the appropriate time here “on the continent”. The picture above is of the Leonard Wolfson Experimental Neurology Centre in London; it is part of UCL and closely affiliated with the National Hospital for Neurology and Neurosurgery.

The PhD interview I was invited to attend for the Wolfson Centre had to be conducted via Skype since I had already flown home, and it was a significantly different experience from the first interviews I had had two weeks previously.

Firstly, the interview panel this time consisted of five group leaders (compared to four), including Nicholas Wood, the professor who runs the programme, Anthony Schapira, the head of the Department of Clinical Neurosciences, and Linda Greensmith, a professor of neuroscience who researches motor neuron disorders. Secondly, since the Skype connection was not perfect it was extremely difficult to see all of them at once, let alone discern their facial expressions. I certainly did not manage to make them laugh once during the 15-minute grilling. Thirdly, and possibly most importantly, there was no paper we had to read beforehand or any presentation we needed to give. This, combined with the fact that I know almost nothing about neuroscience, caused me to be a lot less sure of my abilities. Fourthly, some of the questions they asked were really quite hard. Apart from the more obvious, “Why do you want to join this programme?”, there was also, “What do you think is the role of industry in science?”. To answer the latter question I rambled on a bit about pharmaceutical companies and cancer combination therapies, which is probably not what they really wanted to hear. And then there was also, “What do you consider the most important advancement in biology in the last ten years?”. Luckily, this is something I had thought about during the course of exam preparations in previous years, and so I answered “fluorescent proteins” with quite some confidence and then went on to say that I didn’t think I’d recently read a molecular/cell biology paper that did not utilise fluorescent proteins. Lastly, they understandably also wanted to know what field of neuroscience/neurodegenerative disease research I was particularly interested in. Given that I have never taken a dedicated neuroscience course this was particularly tricky to answer. Thankfully, a lot of the group leaders’ research at UCL focuses on various aspects of mitochondrial (dys)function and so I tried to argue that it would be interesting to find out more about the differences and similarities between mitochondrial functions in various neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases.

Don’t ask me how, but I managed to convince them to let me visit the institute in January so that we can have a proper conversation and more in-depth discussion of the available research projects.

To end on a festive and fluorescent note, here is a picture (taken from the website of Roger Tsien’s lab) of glowing bacteria producing red and green proteins – merry Christmas!

IMAGE - Wreath 2001