CRISPR ecology and evolution

Fighting viruses is no easy task. Bacterial cells have survived phage attacks by evolving sophisticated defence strategies that enable them to thrive even in virus-rich ecosystems. CRISPR-Cas is one of these mechanisms used by microbes to protect against viral infection. Bacterial CRISPR-Cas type II systems function by first incorporating short DNA ‘spacers’, derived from invading defective phage genomes, in the CRISPR array located in their genome. The bacterial CRISPR array is then transcribed and matured into short RNAs, which, by recruiting Cas9 endonuclease, act as surveillance complexes that recognise and cleave subsequent invading matching DNA sequences. The cleavage occurs near a short motif, called the PAM, adjacent to the sequence targeted by the spacer. Phages have evolved counter-tactics to thwart such mechanisms, leading to a so-called biological arms race. For example, phages can bypass CRISPR immunity through point mutation or deletion of the CRISPR target or PAM in their genome as well as by the production of anti-CRISPR proteins (ACRs). Using the Gram-positive dairy bacterium Streptococcus thermophilus as a model, this presentation will recall the roles played by virulent phages in the understanding of CRISPR-Cas systems as well as in the development of industrially relevant phage-resistant bacterial strains. Professor Moineau will also highlight the recent discovery, characterisation and effectiveness of two families of ACRs from S thermophilus phages. The emergence of ACR-containing phages illustrates the ongoing battle between phages and their hosts and that additional approaches are needed to control phages in industrial settings.

Professor Sylvain Moineau, Université Laval, Canada

Professor Sylvain Moineau, Université Laval, Canada

Sylvain Moineau is a Professor in the Department of Biochemistry, Microbiology and Bioinformatics at the Université Laval. He also holds the Canada Research Chair in Bacteriophages and is the Curator of the Félix d’Hérelle Reference Center for Bacterial Viruses, the world largest collection of reference phages. His research group studies phage biology and phage-bacteria interactions. Among others, he has characterized mechanisms used by bacteria to resist phages, including his landmark work on CRISPR-Cas systems. Professor Moineau has won several teaching and research awards. In 2017, he was awarded the NSERC John C. Polanyi Award, the Canadian Society of Microbiologists Murray Award for Career Achievement and the DuPont Excellence Medal. He was elected to the Academy of Sciences of the Royal Society of Canada and appointed an Officer of the Order of Canada. Thomson Reuters and Clarivate Analytics have ranked him amongst the most cited microbiologists for the past four years.

Bacterial CRISPR-Cas systems utilise sequence-specific RNA-guided nucleases to defend against bacteriophage (phage) infection. As a counter-measure, numerous phages produce proteins to block the function of the Class 1 CRISPR-Cas systems, which utilise multi-subunit protein complexes to enact immunity. Dr Bondy-Denomy’s group recently developed and utilised novel bioinformatics strategies to identify Class 2 (eg Cas9 and Cas12) anti-CRISPR proteins. The researchers have identified anti-CRISPR proteins encoded by phages and mobile genetic elements from many organisms, including Pseudomonas aeruginosa, Listeria monocytogenes, Moraxella bovoculi and Streptococcus pyogenes, suggesting widespread and common CRISPR-Cas inactivation. More recently, we have identified a phage with a novel mechanism of CRISPR evasion that does not rely on anti-CRISPR proteins, but instead physically segregates its genome from nucleases. Dr Bondy-Denomy will discuss our progress towards understanding these mechanisms that phages deploy to avoid destruction by CRISPR-Cas and how this work benefits CRISPR-Cas technologies.

Dr Joe Bondy-Denomy, UC San Francisco, USA

Dr Joe Bondy-Denomy, UC San Francisco, USA

Joe attended the University of Waterloo for his undergrad in Biology and Microbiology, and spent time as a co-op student in labs at Western University (London, ON) and McGill University (Montreal, QC). Joe then attended the University of Toronto to complete his PhD in Alan Davidson's lab where he studied interactions between bacteriophages and their host bacteria, focusing on the effects of lysogeny and the CRISPR-Cas immune system. In the spring of 2015, Joe started his lab at UCSF as a Faculty Fellow. In 2017, Joe continued at UCSF as an Assistant Professor in the Department of Microbiology & Immunology. The lab is currently focused on studying the roles of CRISPR-Cas immune systems in their host organisms, discovering novel CRISPR-Cas functions and identifying ways in which these systems interact with bacteriophages.

CRISPR-Cas is an adaptive prokaryotic immune system that prevents phage infection. By incorporating phage-derived “spacer” sequences into CRISPR loci on the host genome, future infections from the same phage genotype can be recognised and the phage genome cleaved. However, phage can escape CRISPR degradation by mutating the sequence targeted by the spacer, allowing them to re-infect previously CRISPR-immune hosts, and theoretically leading to co-evolution. Previous studies have shown that phage can persist over long periods in populations of Streptococcus thermophilus that can acquire CRISPR-Cas immunity, but it has remained less clear whether this coexistence was due to co-evolution, and if so, what type of co-evolutionary dynamics were involved. In this talk Dr van Houte will discuss results from highly replicated serial transfer experiments her group performed over 30 days with S thermophilus and a lytic phage. Using a combination of phenotypic and genotypic data, the researchers show that CRISPR-mediated resistance and phage infectivity coevolved over time following an arms race dynamic, and that asymmetry between phage infectivity and host resistance within this system eventually causes phage extinction. This work provides further insight into the way CRISPR-Cas systems shape the population and co-evolutionary dynamics of bacteria-phage interactions.

Dr Stineke van Houte, University of Exeter, UK

Dr Stineke van Houte, University of Exeter, UK

Stineke van Houte is a microbiologist at the University of Exeter interested in integrating molecular and evolutionary approaches to study host-parasite interactions. To do this she has used a variety of different model systems, including bacteria and phages. Over the past few years she has studied ecological factors that determine the evolution of CRISPR immunity, the role of CRISPR diversity in limiting phage epidemics, and how anti-CRISPR phages can work together to successfully overcome CRISPR immunity in bacteria. She has recently received a BBSRC fellowship and started her own research group, where she studies competition between mobile genetic elements in microbial communities and in particular the role of mobile CRISPR systems therein, and how this knowledge can be used to tackle antimicrobial resistance.

The ecological and evolutionary effects CRISPR-Cas immune system are traditionally studied in controlled laboratory conditions. Aquaculture provides semi-natural conditions for examining the role of CRISPR-Cas in phage/bacterium co-evolution in the environment, as the disease surveillance projects together with interest towards phage therapy have led to collections of phage and bacterial isolates. The researchers sampled a flow-through aquaculture setting during 2007-2014, and characterized the co-evolution of populations of phages and their bacterial hosts Flavobacterium columnare at the phenotypic and genetic level. Bacteria were generally resistant to phages from the past and susceptible to phages isolated in years after bacterial isolation. Two CRISPR loci in the bacterial host were identified: a type II-C locus (with Cas9) and a type VI-B locus (with Cas13a). Phage-matching spacers appeared in both loci over time. The spacers mostly targeted the terminal end of the phage genomes, which also varied across time, resulting in arms-race-like changes in the protospacers of the coevolving phage population. On several occasions, the introduction of CRISPR spacers to the bacterial population was followed by the appearance of phage isolates with modifications in the corresponding protospacer regions. The researchers’ results demonstrate that aquaculture can provide essential information on the co-evolutionary dynamics between the phage and its host under environmental conditions.

Dr Lotta-Riina Sundberg, University of Jyväskylä, Finland

Dr Lotta-Riina Sundberg, University of Jyväskylä, Finland

Dr Sundberg is a senior lecturer and a PI at the Department of Biological and Environmental Science and Nanoscience Center in University of Jyväskylä, Finland. During 2013-2018 she held the Academy Researcher position in the Centre of Excellence in Biological Interactions, and focused on multirophic coevolutionary dynamics of metazoan-bacterium and bacterium-phage interactions. More recently, Sundberg has studied phage evolution and bacterial CRISPR immunity in freshwater systems and in settings relevant to intensive aquaculture. For example, her studies on the phage-bacterium coevolution in the fish pathogen Flavobacterium columnare have shown that the coevolutionary arms race in the environment selects for increased host range and infectivity in the phage. Molecular understanding of these interactions in aquaculture brings insight also on the development of phage therapy.

The emergence of pathogens remains a major public health concern. Unfortunately, when and where pathogens will (re-)emerge is notoriously difficult to predict as the erratic nature of those events is reinforced by the stochastic nature of pathogen evolution during the early phase of an epidemic. For instance, mutations allowing pathogens to escape host resistance may boost pathogen spread and promote emergence. Yet the ecological factors that govern such evolutionary emergence remain elusive both because of the lack of ecological realism of current theoretical frameworks and the difficulty of experimentally testing their predictions. The researchers developed a theoretical model to explore the effects of the heterogeneity of the host population on the probability of pathogen emergence, with or without pathogen evolution. The researchers show that evolutionary emergence and the spread of escape mutations in the pathogen population is more likely to occur when the host population contains an intermediate proportion of resistant hosts. This probability of evolutionary emergences rapidly declines with the diversity of resistance in the host population but also with the multiplicity of host resistance per individual host. Experimental tests using lytic bacteriophages infecting their bacterial hosts containing CRISPR-Cas immune defenses confirm these theoretical predictions. Dr Gandon will discuss how these results can help design more effective control strategies against the emergence of infectious diseases.

Dr Sylvain Gandon, CNRS Montpellier, France

Dr Sylvain Gandon, CNRS Montpellier, France

I am an evolutionary biologist with a broad general interest ranging from theoretical population genetics, and mathematical epidemiology, to behavioral and evolutionary ecology. I use both theoretical and experimental approaches to study the ecology and evolution of host-parasite interactions. I studied the influence of spatial structure and migration on host-parasite coevolution and local adaptation. I am also interested in the impact of therapeutic interventions like drugs or vaccines on the evolution of pathogens. I am currently working on two experimental models: (1) the coevolution between bacteria and their bacteriophages and (2) the evolutionary ecology of avian malaria.

Many archaea possess spacers that match chromosomal genes of related species, including those encoding core housekeeping genes. By sequencing genomes of environmental archaea isolated from a single site, Dr Gophna’s group demonstrated that inter-species spacers are common. They then showed experimentally, by mating Haloferax volcanii and Haloferax mediterranei, that spacers are indeed acquired chromosome-wide, although a preference for integrated mobile elements and nearby regions of the chromosome exists. Engineering the chromosome of one species to be targeted by the other’s CRISPR–Cas reduces gene exchange between them substantially. Thus, spacers acquired during inter-species mating could limit future gene transfer, resulting in a role for CRISPR–Cas systems in microbial speciation. More recently the researchers have identified a virus that chronically infects one of their natural Haloferax isolates and can also integrate into its genome. Exposure to this virus elicited strong and specific spacer acquisition by the H volcanii lab strain, that surprisingly could not be stably infected by that virus. This raised the question why the virus' original host that appears to have a functional CRISPR-Cas system did not acquire spacers from a virus that chronically infects it. The researchers addressed this by an “active immunisation” approach in which provirus-containing cells were exposed to mature virus particles. K Höveler, J Deiglmayr and M Beyer from Physical Chemistry Laboratory, ETH Zurich, Switzerland also contributed to the research outlined in this talk.

Dr Uri Gophna, Molecular Cell Biology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Israel

Dr Uri Gophna, Molecular Cell Biology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Israel

Professor Uri Gophna’s research is focused on understanding the evolutionary processes behind microbial adaptation that has contributed to both the medical community and to evolutionary theory. His research group’s interests revolve around two related topics: the role of lateral gene transfer (LGT) and selfish DNA in the evolution of microorganisms and the study of host-microbe interactions, with particular focus on the human gut microbiome in health and disease.

Their work spans the whole spectrum of evolutionary biology from environmental and comparative population genomics all the way to the testing of specific hypothesis by engineering strains and performing competition experiments under ecologically relevant conditions. Genetic manipulation allows the dissection of the contribution of specific genetic components, such as defense systems (CRISPR-Cas and additional recently discovered systems), the recombination machinery, and selfish mobile DNA, to genome diversity within microbial populations.

In this talk Professor Burt will describe how CRISPR technology may be used to develop new tools for controlling disease vectors and other pest species. The primary focus will be on a particular case study, the development of gene drive approaches to control mosquitoes that transmit malaria in sub-Saharan Africa. Gene drive is a naturally occurring process by which genes are able to bias their transmission from one generation to the next, and can allow a gene to spread through a population even if it is harmful to the organisms carrying it. Synthetic gene drive systems have been developed in mosquitoes using CRISPR technology and proof-of-principle for population control demonstrated in the lab. Issues arising in the further development of this technology will be discussed, and then prospects for CRISPR-based tools to control other pest species.

Professor Austin Burt, Imperial College London, UK

Professor Austin Burt, Imperial College London, UK

Austin Burt is Professor of Evolutionary Genetics at Imperial College London. In 2003 he proposed that enzymes that recognise and cut specific sequences of DNA could be used to control pest populations, and since 2005 he has been Principle Investigator of a research consortium developing this idea to control the mosquitoes that transmit malaria, with particular attention to sub-Saharan Africa. Target Malaria is a not-for-profit consortium that currently includes over 100 individuals at 14 institutions across 3 continents (www.targetmalaria.org).

In recent years, new genome editing technologies have emerged that can edit the genome of non-human animals with progressively increasing efficiency. Despite ongoing academic debate about the ethical implications of these technologies, no comprehensive overview of this debate exists. To address this gap in the literature, the researchers conducted a systematic review of the reasons reported in the academic literature for and against the development and use of genome editing technologies in animals. Most included articles were written by academics from the biomedical or animal sciences. The reported reasons related to seven themes: human health, efficiency, risks and uncertainty, animal welfare, animal dignity, environmental considerations and public acceptability. The findings illuminate several important considerations about the academic debate, including a relatively low disciplinary diversity in the contributing academics, a scarcity of systematic comparisons of potential consequences of using these technologies, an underrepresentation of animal interests, and a disjunction between the public and academic debate on this topic. As such, this article can be considered a call for a broad range of academics to get increasingly involved in the discussion about genome editing, to incorporate animal interests and systematic comparisons, and to further discuss the aims and methods of public involvement.

Dr Sarah Hartley, Exeter University, UK

Dr Sarah Hartley, Exeter University, UK

Nienke de Graeff, University Medical Center Utrecht, the Netherlands

Nienke de Graeff, University Medical Center Utrecht, the Netherlands

Nienke de Graeff is a PhD Candidate in the ethics of biomedical innovation at the department of Medical Humanities at the University Medical Center Utrecht. She conducts research in the field of bioethics and genome-editing technologies. In her PhD project, she focuses on the ethical analysis of gene drives. She works in the group of Professor Dr Annelien Bredenoord, which engages in so-called ‘ethics parallel research’, in which ethicists collaborate with scientists from the earliest stages of research to identify and evaluate ethical issues of novel technologies proactively as the field develops.

Nienke has a background in the Liberal Arts & Sciences (BSc University College Maastricht), Medicine (MSc Selective Utrecht Medical Master Utrecht) and Applied Ethics (MA Applied Ethics, Utrecht University).