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

CRISPR Digest #5

Edit on April 23rd 2015: Just three days after posting about “a prudent path forward for genomic engineering” I read this Nature News & Comment article describing the experiments of a research group in Guangzhou, China in which they used CRISPR/Cas9 technology on human embryos (Liang et al. (2015)). [Their article was published open access in Protein Cell and can be found here.] Admittedly, the embryos they used came from so-called tripronuclear zygotes, which are zygotes formed when an egg cell is fertilised by two sperm cells as can happen during in vitro fertilisation. These embryos are therefore not viable in vivo. However, the question remains whether this is “prudent” use of CRISPR technology. On the one hand, yes, if genome-editing in humans is ever going to happen on a regular basis then we need to understand exactly how it works. For instance, they do show that their approach has considerable off-target effects: their guide RNAs were targeting the beta-globin gene (which encodes a subunit of haemoglobin) but DNA breaks were also induced at other sites. On the other hand, experimenting on human embryos will (probably) always be scrutinised with ethical concerns in mind, especially because it is really not clear that genome-editing in human embryos should be the way forward.


Since the last CRISPR update there have been some developments regarding this new genome-editing technique. Leading scientists in the field (Baltimore et al (2015)) met in Napa, California at a bioethics conference organised by the Innovative Genomics Initiative (IGI) to discuss CRISPR policy and make discussion of this topic more visible to and inclusive of doctors, social scientists and the public. They pinpoint four recommendations to be put into immediate action:

  1. Strong discouragement of “any attempts at germline genome modification for clinical application in humans, while societal, environmental, and ethical implications of such activity are discussed among scientific and governmental organizations”.
  2. Creation of forums of experts in science and ethics to discuss the potentials and risks of this technology.
  3. Transparent research to gain a better understanding of how CRISPR works.
  4. Creation of a “globally representative group of developers and users of genome engineering technology […] to recommend policies”.

I noticed that Feng Zhang of MIT was not a co-author of this paper (and neither was Emmanuelle Charpentier, to my surprise), but maybe this is because he is now or will soon be involved in a “winner-take-all” patent dispute. At the moment, Zhang (and MIT/Broad Institute?) own the rights to CRISPR and claim that they invented the technology first/were the first to make it work. However, their patent application was filed half a year after the Doudna/Charpentier patent in 2012. So now the University of California in Berkeley (where Doudna works) and the University of Vienna (where Charpentier worked) set up a so-called patent interference request and if they are successful they will own CRISPR rights entirely, leaving Zhang with nothing. The whole process is summarised here, from where I also copied this figure, showing the number of papers published on CRISPR in the past ten years:

crispr paper numbers

The graph illustrates how much is at stake during this patent interference process. Equally, the technology is getting attention in non-scientific circles: Time Magazine’s 100 most influential people include Jennifer Doudna (left) and Emmanuelle Charpentier (right) in the “pioneer” category. Here they are last year at the Breakthrough Prize in Life Sciences ceremony probably looking very much the opposite of what society at large thinks scientists (should) look like:

Life Sciences co-laureates Doudna and Charpentier speak on stage during the 2nd Annual Breakthrough Prize Awards in Mountain View

References:

Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M, Greely HT, Jinek M, Martin GS, Penhoet E, Puck J, Sternberg SH, Weissman JS, Yamamoto KR (2015) Biotechnology. A prudent path forward for genomic engineering and germline gene modification. Science 348: 36-38

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

CRISPR Digest #3

First post of the new year, so it only makes sense to go back to one of my favourite topics: CRISPR. As you can imagine, the recent breakthrough in applying the CRISPR technology and not merely using it in basic research has sparked a large patent dispute. Although Jennifer Doudna and Emmanuelle Charpentier originally recognised the method’s potential, it is Feng Zhang who won a large patent over the technology last year (discussed in detail here). However, as far as I understand it, a legal process will be launched in which it may still be possible to reverse the patent ownership. And this would have implications for the various biotechnology start-ups, including Editas Medicine (founded by Zhang, Doudna and others; although Doudna is no longer involved with this company) and CRISPR Therapeutics (founded by Charpentier and others), that were recently launched to utilise CRISPR in therapeutic approaches. I agree with Charpentier when she says that “things are happening fast, maybe a bit too fast”, but she also seems confident that “the story is going to end up well”. According to either John Lennon, Fernando Sabino or an Indian proverb “In the end, everything will be okay. If it’s not okay, it’s not yet the end.” That must be a comforting thought when many millions of dollars are at stake.

But it is not only biotechnology start-ups that have a vested interested in CRISPR technologies. For example, the pharmaceutical company Novartis has recently released information about its attempts to use genome-editing to increase the efficacy of immunotherapy approaches – the use of immune cells, such as “killer T cells”, to treat cancers by instructing them to target malignant cells – in the treatment of haematological cancers.

Following on from the last digest (Hu et al. (2014)), more research groups are using CRISPR to investigate the interactions between HIV and human cells. In one paper (Wang et al. (2014)) they managed to disrupt CCR5, one of the main cell surface receptors of HIV-1, leading to the generation of HIV-resistant cells.

Lastly, CRISPR is being adopted as a tool by researchers in ever more diverse fields of study. For example, in addition to crop plants and HIV, a very recent paper (Peng et al. (2015)) shows the use of CRISPR in Trypanosoma cruzi, the causative agent of human Chagas disease, and a relative of Trypanosoma brucei, the causative agent of human sleeping sickness and nagana in cattle. Yet another group (Kistler et al. (2014)) used CRISPR to engineer the genome of the mosquito Aedes aegypti, the transmitter of yellow fever and dengue viruses. These advances, although at the moment “only” representing proof-of-principle experiments, may in future lead to more effective methods of interfering with the virulence of these protozoan parasites and virus vectors (both images from the Centers for  Disease Control and Prevention):

Untitled

References:

Hu WH, Kaminski R, Yang F, Zhang YG, Cosentino L, Li F, Luo BA, Alvarez-Carbonell D, Garcia-Mesa Y, Karn J, Mo XM, Khalili K (2014) RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection.Proceedings of the National Academy of Sciences of the United States of America 111: 11461-11466

Kistler KE, Vosshall LB, Matthews BJ (2014) Genome-engineering with CRISPR-Cas9 in the mosquito Aedes aegypti. 

Peng D, Kurup SP, Yao PY, Minning TA, Tarleton RL (2015) CRISPR-Cas9-Mediated Single-Gene and Gene Family Disruption in Trypanosoma cruzi. mBio 6

Wang W, Ye C, Liu J, Zhang D, Kimata JT, et al. (2014) CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection. PLoS ONE 9(12): e115987.